We thank Veronica Schreiter for her excellent technical assistance and helpful modifications of the established cryopreservation technique and cell culture model. Additionally, we like to thank Maren Anders and Swanhild Rodewald for their excellent technical assistance in the subsequent analysis.

NOTE: Bovine ovaries were obtained from a commercial slaughterhouse. The collection of abattoir byproducts does not require an ethical approval according to the German law.
1. Working Conditions and Preparations
<ol>
	<li>To guarantee sterility, perform all media and tissue preparation as well as all culture work in a specialized cell culture lab using a laminar flow bench.</li>
	<li>Prepare 1x phosphate buffered saline (PBS, pH 7.4) supplemented with 100 IU penicillin, 0.1 mg/mL streptomycin and 0.5 &#181;g/&#181;L amphotericin for the transport of ovaries. 
	NOTE: The maximal duration between receiving the ovaries and isolating the GC is 2 h in this set-up.</li>
</ol>
2. Isolation of Bovine Granulosa Cells
<ol>
	<li>Wash the ovaries several times in 1x PBS (with 100 IU penicillin, 0.1 mg/mL streptomycin, and 0.5 &#181;g/&#181;L amphotericin) to remove any blood from the surface before starting the isolation procedure. Use a beaker, place the ovaries inside, fill it with 1x PBS, and discard the PBS again.
	<ol>
		<li>Repeat this washing step 3&#8211;4x, until the ovaries are cleaned from any remaining blood.</li>
	</ol>
	</li>
	<li>Wipe one ovary using a lab wipe soaked with 70% alcohol, to minimize possible contaminations.</li>
	<li>With a 3 mL syringe and an 18 G needle, aspirate the GC by puncturing small- to medium-sized follicles (&#60;6 mm, measured with a ruler), and pool the follicular fluid in one 50 mL centrifuge tube. 
	NOTE: To moisten the syringe, take a little amount of 1x PBS (with 100 IU penicillin, 0.1 mg/mL streptomycin, and 0.5 &#181;g/&#181;L amphotericin). This helps to collect small amounts of follicular fluid and prevents cells from sticking inside the syringe.</li>
	<li>After puncturing several follicles, rinse the syringe and needle with 1x PBS for intermediate cleaning.</li>
</ol>
3. Cryopreservation of Cells
<ol>
	<li>Use trypan blue staining and count the cells under a hemocytometer.
	<ol>
		<li>To count the number of viable cells, prepare a tube with 1.5 &#181;L of a 0.25% trypan blue solution. 
		NOTE: The living cells will remain unstained because trypan blue can only access the cell barrier of dead cells, which then appear blue.</li>
		<li>Add 15 &#181;L of the granulosa cell suspension to the trypan blue solution and mix them gently.</li>
		<li>Place the mixture in both chambers of a hemocytometer. Count the number of living (e.g., unstained) cells in one big square per chamber. 
		NOTE: Although some blood cells can be seen, they can be distinguished by their very small size.</li>
		<li>Calculate the number of living cells with the mean of both chamber squares according to the general guidelines; the proportion of living cells can vary between ovaries but should exceed 60%13.</li>
	</ol>
	</li>
	<li>Take a sample from the freshly isolated granulosa cell pool as a reference for further analysis.
	<ol>
		<li>Depending on the cell count of the isolated GC, set aside an appropriate number of cells as reference samples for RNA, DNA, or protein preparation. 
		NOTE: For RNA isolation and the subsequent evaluation of the gene expression, 1 x 105 cells are sufficient, whereas other subsequent analyses might require different, generally higher cell numbers.</li>
		<li>Place one or several aliquots of the reference cells in a fresh tube and centrifuge it for 1 min at room temperature and maximum speed (12,000 x g) to obtain a cell pellet. Discard the supernatant.</li>
		<li>Immediately shock-freeze the samples in liquid nitrogen. Store the sample at -80&#160;&#176;C.</li>
	</ol>
	</li>
	<li>Perform the cryopreservation as follows.
	<ol>
		<li>Prepare the freezing medium using 80% fetal calf serum (FCS) and 20% dimethyl sulfoxide (DMSO).</li>
		<li>In order to gently pellet the GC, centrifuge the GC for 3 min at room temperature and 500 x g.</li>
		<li>Remove the supernatant carefully and gently resuspend the cells in 100% FCS (e.g., 1 mL) until no more cell clumps are visible.</li>
		<li>Add 1 volume (e.g., 1 mL) of the prepared freezing medium to the cell suspension and transfer the mixture to a cryogenic vial. 
		NOTE: The final concentration of the cryo-protecting media will be 90% FCS and 10% DMSO.</li>
		<li>Place the cryogenic vial in a specialized (commercially available) freezing container that prevents rapid freezing. The cooling rate of the samples should not exceed -1&#160;&#176;C/min. Transfer the freezing container into a -80&#160;&#176;C freezer for at least 4 h.</li>
		<li>For longer storage, place the cryogenic vials in ultra-low temperature containers (e.g., liquid phase nitrogen containers). 
		NOTE: The protocol can be stopped here, as the cryopreservation allows longer storage.</li>
	</ol>
	</li>
</ol>
4. Preparation of Media and Culture Plates for the Cell Culture
<ol>
	<li>Coat cell culture plates with 0.02% collagen R. 
	NOTE: Collagen R is known to be essential for an improved attachment of GC in culture.
	<ol>
		<li>Prepare a 0.02% collagen R solution with purified water suitable for cell culture.</li>
		<li>Add 150 &#181;L per well in a 24-well plate (= 2 cm2/well) and ensure that the whole surface of the culture well is covered with the collagen R solution.</li>
		<li>Let the solution dry with the lid open for a few hours or overnight in the laminar flow hood.</li>
		<li>When the culture plates are completely dry, irradiate them with UV light for 10&#8211;15 min to minimize contamination.</li>
		<li>If necessary, store the plates at 4&#160;&#176;C for up to 2 months.</li>
	</ol>
	</li>
	<li>Prepare media and supplements for culture work under a laminar flow hood. For a successful recovery of the GC, they have to be processed immediately after thawing.</li>
	<li>Prepare the following media.
	<ol>
		<li>Supplement &#945;-Minimal Essential Medium (&#945;-MEM) with 2 mM L-glutamine, 0.084% sodium bicarbonate, 0.1% bovine serum albumin (BSA), 20 mM HEPES, 4 ng/mL sodium selenite, 5 &#181;g/mL transferrin, 10 ng/mL insulin and 1 mM non-essential amino acids.</li>
		<li>Additionally, add 100 IU penicillin and 0.1 mg/mL streptomycin to the media. 
		NOTE: The antibiotics, once warmed, are stable for approximately 48 h. Therefore, aliquot the desired amount of media for the culture set-up and planned media exchange. Pre-warm only the media aliquots to 37&#160;&#176;C in a water bath. The prepared media can be stored at 4&#160;&#176;C for no longer than 3 months.</li>
		<li>To induce steroid activity in cultured bovine GC, supplement the &#945;-MEM additionally with 20 ng/mL follicle-stimulating hormone (FSH), 50 ng/mL R3 IGF-1, and 2 &#181;M androstenedione. 
		NOTE: Always supplement these components shortly before the onset of a culture or media exchange. Avoid repeated freeze-and-thaw cycles for FSH and IGF-1 stock solutions, as this might compromise the steroid activity.</li>
	</ol>
	</li>
</ol>
5. Cell Culture Work
<ol>
	<li>Thaw the cells quickly in a water bath at 37&#160;&#176;C for 3&#8211;4 min and transfer the cell suspension into 37&#160;&#176;C pre-warmed &#945;-MEM (without hormone supplement). For centrifugation, a final volume of 10 mL is recommended.</li>
	<li>Centrifuge the GC for 3 min at room temperature and at 500 x g. Discard the supernatant. Add the pre-warmed and supplemented &#945;-MEM and resuspend the cells carefully.</li>
	<li>Seed the GC at a density of 1 x 105 or 1 x 106 cells per well in a final volume of 500 &#181;L. Include technical replicates of at least three culture wells per condition.</li>
	<li>Incubate the cell culture in an incubator at 37&#160;&#176;C with 5% CO2 for 8 d.</li>
	<li>Perform a media exchange every other day. Replace only two-thirds of the culture media to reduce stress and to ease the adaptation of the cells to the fresh media. 
	NOTE: The GC form a typical fibroblast-like phenotype after a few days in culture and tend to cluster together. In the high cell-density culture, larger clusters are found more frequently as compared to the normal density GC culture (Figure 1, left vs. right panel).</li>
</ol>
6. Subsequent Analysis of Cultured Granulosa Cells
<ol>
	<li>To perform steroid hormone analysis, collect the media and freeze it at -20&#160;&#176;C. Use appropriate methods to analyze the estradiol and progesterone concentration in the spent media [e.g., radioimmunoassay (RIA)]14.</li>
	<li>For the subsequent analysis of different target molecules (RNA, DNA, proteins, etc.), lyse the cells directly in the culture dish with an appropriate lysing agent as recommended by the supplier. 
	NOTE: For most isolation procedures, it is possible to stop the protocol here, as the lysed cells can be stored at -20&#160;&#176;C for up to 1 week. For longer storage, the cells should be kept at -80&#160;&#176;C.</li>
	<li>To determine the transcript abundance, synthesize cDNA and measure specific transcripts by quantitative real-time polymerase chain reaction (qPCR) techniques15. A statistical evaluation should include at least three replications of different cell cultures, originated from different cell preparations (biological replicates).</li>
</ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+heat+stress+on+the+survival+and+development+of+in+vitro+cultured+bovine+preantral+follicles+and+on+in+vitro+maturation+of+cumulus-oocyte+complex.">Effect of heat stress on the survival and development of in vitro cultured bovine preantral follicles and on in vitro maturation of cumulus-oocyte complex.</a> Theriogenology. 86 (4), 994-1003 (2016).</li><li>Christenson, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+resource:+preovulatory+LH+surge+effects+on+follicular+theca+and+granulosa+transcriptomes.">Research resource: preovulatory LH surge effects on follicular theca and granulosa transcriptomes.</a> Molecular Endocrinology. 27 (7), 1153-1171 (2013).</li><li>Havelock, J. C., Rainey, W. E., Carr, B. 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J., Webb, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+maintenance+of+oestradiol+and+immunoreactive+inhibin+production+with+FSH+by+ovine+granulosa+cells+cultured+in+serum-free+media.">Induction and maintenance of oestradiol and immunoreactive inhibin production with FSH by ovine granulosa cells cultured in serum-free media.</a> Journal of Reproduction and Fertility. 106 (1), 7-16 (1996).</li><li>Hillier, S. G., Whitelaw, P. F., Smyth, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicular+Oestrogen+Synthesis+-+The+Two-Cell,+Two-+Gonadotrophin+Model+Revisited.">Follicular Oestrogen Synthesis - The Two-Cell, Two- Gonadotrophin Model Revisited.</a> Molecular and Cellular Endocrinology. 100, 51-54 (1994).</li><li>Silva, J. M., Price, C. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+plating+density+alters+the+ratio+of+estrogenic+to+progestagenic+enzyme+gene+expression+in+cultured+granulosa+cells.">Cell plating density alters the ratio of estrogenic to progestagenic enzyme gene expression in cultured granulosa cells.</a> Fertility and Sterility. 93 (6), 2050-2055 (2010).</li><li>Baufeld, A., Vanselow, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+cell+plating+density+mimics+an+early+post-LH+stage+in+cultured+bovine+granulosa+cells.">Increasing cell plating density mimics an early post-LH stage in cultured bovine granulosa cells.</a> Cell and Tissue Research. 354, 869-880 (2013).</li><li>Schneider, F., Br&#252;ssow, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+a+preovulatory+administered+depot+gonadotrophin-releasing+hormone+agonist+on+reproductive+hormone+levels+and+pregnancy+outcome+in+gilts.">Effects of a preovulatory administered depot gonadotrophin-releasing hormone agonist on reproductive hormone levels and pregnancy outcome in gilts.</a> Reproduction, Fertility and Development. 18 (8), 857-866 (2006).</li><li>Baufeld, A., Koczan, D., Vanselow, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+altered+gene+expression+profiles+in+cultured+bovine+granulosa+cells+at+high+cell+density.">Induction of altered gene expression profiles in cultured bovine granulosa cells at high cell density.</a> Reproductive Biology and Endocrinology. 15 (1), (2017).</li><li>Baufeld, A., Vanselow, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactate+promotes+specific+differentiation+in+bovine+granulosa+cells+depending+on+lactate+uptake+thus+mimicking+an+early+post-LH+stage.">Lactate promotes specific differentiation in bovine granulosa cells depending on lactate uptake thus mimicking an early post-LH stage.</a> Reproductive Biology and Endocrinology. 16 (1), 15 (2018).</li><li>Yenuganti, V. R., Vanselow, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultured+bovine+granulosa+cells+rapidly+lose+important+features+of+their+identity+and+functionality+but+partially+recover+under+long-term+culture+conditions.">Cultured bovine granulosa cells rapidly lose important features of their identity and functionality but partially recover under long-term culture conditions.</a> Cell and Tissue Research. 368 (2), 397-403 (2017).</li><li>Hamel, M., Vanselow, J., Nicola, E. S., Price, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Androstenedione+Increases+Cytochrome+P450+Aromatase+Messenger+Ribonucleic+Acid+Transcripts+in+Non-Luteinizing+Bovine+Granulosa+Cells.">Androstenedione Increases Cytochrome P450 Aromatase Messenger Ribonucleic Acid Transcripts in Non-Luteinizing Bovine Granulosa Cells.</a> Molecular Reproduction and Development. 70, 175-183 (2005).</li><li>Gilbert, I., Robert, C., Dieleman, S., Blondin, P., Sirard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+effect+of+the+LH+surge+in+bovine+granulosa+cells+during+the+peri-ovulation+period.">Transcriptional effect of the LH surge in bovine granulosa cells during the peri-ovulation period.</a> Reproduction. 141 (2), 193-205 (2011).</li></ol>

The authors have nothing to disclose.

The presented cell culture model provides a tool to analyze granulosa cell differentiation in vitro. Several studies showed that a serum-free cultivation is a prerequisite to maintaining steroid activity in cultured bovine GC or GC of other species8,9. Additionally, coating the culture dish with components of the extracellular matrix (e.g., collagen R)13, improved the attachment of the cells significantly. Another important feature is the prolonged culture period. Recently, it has been demonstrated that a long-term culture is necessary to obtain sufficient steroidogenic activity and a balanced expression of granulosa cell identity markers17. It appears that GC require the time to recover from the physical stress during the isolation procedure.
The media supplements FSH, IGF-1, and androstenedione are known to induce aromatase activity in cultured GC. Especially, the supplementation with androstenedione is absolutely necessary, as the GC need a precursor for estradiol synthesis. This has been published previously11,18 and, therefore, was not further investigated during the present study. However, an adaptation of FSH, IGF-1, and androstenedione concentrations might be necessary for other experimental set-ups.
The cryopreservation technique described here can help to improve the organization of tissue culture experiments by making them more independent from the varying supply with ovaries. According to previous testing, cryopreservation does not affect the GC phenotype or steroid production in culture. Also, the abundance of marker transcripts in cultured cells did not reveal significant differences comparing samples prepared from freshly isolated cells with those previously subjected to cryopreservation16.
A crucial parameter for the present GC culture model is the cell plating density. As shown by the Representative Results, increasing the plating density induced remarkable changes of physiological and molecular characteristics. Several genes are regulated in a specific manner, resembling the changes that are induced by LH stimulation in vivo4,19. The fact that an increasing cell density can drive differentiation-like processes in cultured bovine GC has to be meticulously considered in this GC in vitro model to avoid conflicting results between replicates. Therefore, contradictory results with other studies might be ascribed to different cell densities and should be examined more closely.
The culture model described here revealed to be non-responsive to LH, as the transcripts of the receptor LHCGR are close to the detection limit. Hence, a simulation of the LH surge alike the in vivo situation failed to induce differentiation13. Nonetheless, this model provides a helpful tool to study estradiol-active GC in primary culture, in particular as no functional bovine GC lines exist at present.
Different treatment protocols can be tested in the present GC culture model that help to unravel regulatory mechanisms of steroid production or GC differentiation. Further, single factors that are involved in developmental processes can be separately analyzed. Therefore, this culture model provides a basis for many different applications.

Successful ovulation and luteinization depend on finely tuned and well-orchestrated molecular alterations in different somatic follicular cell types. Since details of these developmental processes are not yet fully understood, further clarification is required. In vivo approaches are elaborate and costly and, in particular, fail to address specific molecular mechanisms that occur during folliculogenesis. Therefore, reductionistic in vitro models are needed in addition, to provide insight into cellular and molecular details. Different studies describe the culture of whole follicles in the context of in vitro fertilization techniques1,2,3. Because researchers are interested in mechanisms of differentiation, many studies focus on follicular GC. These cells, directly associated with the oocyte, are the major sources of estrogen production and, thus, play an essential role throughout folliculogenesis and luteinization4.
Immortalized cell lines of GC have been developed from different species. Most of them, however, do not show a sufficient steroid hormone production5. So far, only one cell line of bovine GC has been established6, but this line lost its steroidogenic activity after several passages7. Therefore, since steroidogenesis and, in particular, estradiol production is an essential feature of GC functionality, it is advisable to study these aspects in primary cell culture models. In previous studies, it was demonstrated that a considerable estradiol production can only be observed under serum-free culture conditions8,9. Further on, the supplementation of a precursor of estradiol synthesis is another prerequisite, as GC fail to express the necessary enzyme that can convert progesterone to androstenedione10. Additionally, the synergistic effect of FSH and IGF-1 supplementation in vitro revealed an optimized activity of aromatase, the key enzyme of estradiol synthesis11. In the present protocol, other important factors that have a substantial impact on the GC culture model are also described. In particular, the cell plating density has tremendous effects on the outcome of the experiment12. Furthermore, a cryopreservation technique of bovine GC that does not significantly interfere with GC physiology in culture could be established. This technique helps to improve the organization of cell culture work and to optimize the preferred plating density.

<table><tbody><tr><td>PBS Instamed w/o Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Merck-Millipore</td><td>L 182-05</td><td>originally purchased at Biochrom AG that is now part of the Merck-Millipore Company; the Cat.-No of Biochrom still exists</td></tr><tr><td>penicillin / streptomycin (10,000 IU / 10,000 &amp;micro;g/mL)</td><td>Merck-Millipore</td><td>A 2213</td><td>originally purchased at Biochrom AG that is now part of the Merck-Millipore Company; the Cat.-No of Biochrom still exists</td></tr><tr><td>amphotericin (250 &amp;micro;g/mL)</td><td>Merck-Millipore</td><td>A 2612</td><td>originally purchased at Biochrom AG that is now part of the Merck-Millipore Company; the Cat.-No of Biochrom still exists</td></tr><tr><td>3 mL syringe (Omnifix Luer)</td><td>Braun</td><td>4616025V</td><td></td></tr><tr><td>18 G needle</td><td>Roth</td><td>C724.1</td><td></td></tr><tr><td>fetal calf serum</td><td>Merck-Millipore</td><td>S 0115</td><td>originally purchased at Biochrom AG that is now part of the Merck-Millipore Company; the Cat.-No of Biochrom still exists</td></tr><tr><td>cryo-preservation vials (2 mL)</td><td>TPP Faust</td><td>TPP 89020</td><td></td></tr><tr><td>CoolCell LX cell freezing container</td><td>Corning</td><td>432004</td><td></td></tr><tr><td>culture dish, 24-well, flat bottom</td><td>TPP Faust</td><td>TPP 92424</td><td></td></tr><tr><td>collagen R (0.2 %)</td><td>Serva</td><td>47254</td><td></td></tr><tr><td>&amp;alpha;-MEM</td><td>Merck-Millipore</td><td>F 0915</td><td>originally purchased at Biochrom AG that is now part of the Merck-Millipore Company; the Cat.-No of Biochrom still exists</td></tr><tr><td>L-Glutamin (200 mM)</td><td>Merck-Millipore</td><td>K 0282</td><td>originally purchased at Biochrom AG that is now part of the Merck-Millipore Company; the Cat.-No of Biochrom still exists</td></tr><tr><td>sodium bicarbonate</td><td>Merck-Millipore</td><td>L 1713</td><td>originally purchased at Biochrom AG that is now part of the Merck-Millipore Company; the Cat.-No of Biochrom still exists</td></tr><tr><td>BSA</td><td>Sigma</td><td>A3311</td><td></td></tr><tr><td>HEPES</td><td>Merck-Millipore</td><td>L 1603</td><td>originally purchased at Biochrom AG that is now part of the Merck-Millipore Company; the Cat.-No of Biochrom still exists</td></tr><tr><td>sodium selenite</td><td>Sigma</td><td>S9133</td><td>dissolved in sterile water</td></tr><tr><td>transferrin</td><td>Sigma</td><td>T1283</td><td>dissolved in sterile water</td></tr><tr><td>insulin (10 mg/mL)</td><td>Sigma</td><td>I0516</td><td>dissolved in 1x PBS</td></tr><tr><td>NEA</td><td>Merck-Millipore</td><td>K 0293</td><td>originally purchased at Biochrom AG that is now part of the Merck-Millipore Company; the Cat.-No of Biochrom still exists</td></tr><tr><td>FSH</td><td>Sigma</td><td>F4021</td><td>we prefer to use a stock solution of 2 &amp;micro;g/mL, diluted in 0.9 % NaCl</td></tr><tr><td>LR3-IGF-1</td><td>Sigma</td><td>I1146</td><td>we use a stock solution of 2 &amp;micro;g/mL, dissolved in 10 mM HCl and 1x PBS with 1 mg/mL BSA</td></tr><tr><td>androstenedione</td><td>Sigma</td><td>46033</td><td>we use a stock solution of 10 mM, diluted in 100 % ethanol</td></tr></tbody></table>

a tissue culture model of estrogen-producing primary bovine granulosa cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

GC plated at 1 x 105 cells/well display a typical fibroblast-like appearance as they form a cell extension comparable to fibroblasts and tend to build clusters (Figure 1a and 1c). Increasing the plating density by 10-fold to 1 x 106 cells/well did not change the morphology but more cell clusters can be observed (Figure 1b and 1d, arrows). As already shown in a previous publication, the collagen coating of the culture dishes largely improved the attachment of the cells13.
Initial cryopreservation did not considerably change the physiological characteristics of cells in culture as compared to those directly cultured from freshly isolated samples. This is shown in Figure 2 as the transcript abundance (measured by qPCR) of several marker genes did not differ when comparing cultured cells derived from either frozen or freshly isolated pools.
Figure 3 shows representative results of the steroid hormone analysis (measured by RIA) in bovine GC cultured at normal vs. high density. The estradiol concentration is significantly decreased in the high-density culture compared to the normal-density culture, whereas the progesterone production tended to be higher.
A comparative analysis of several genes (measured by qPCR) in high- vs. normal-density cultures revealed a significant effect (Figure 4). CYP19A1, encoding the key enzyme of estradiol biosynthesis aromatase, as well as the gonadotropin receptor FSHR, were significantly down-regulated. In contrast, the genes RGS2 and VNN2 showed a significant up-regulation. These results clearly suggest that specific processes of cellular differentiation are induced by increasing the cell plating density.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58208/58208fig1.jpg" /> 
Figure 1: Cultured bovine granulosa cells at normal (left panels) and high cell density (right panels). (a and c) Cells cultured at a density of 1 x 105 cells/well displayed a typical fibroblast-like phenotype. (b and d) GC cultured at a high plating density (1 x 106 cells/well) tended to form large cell clusters (arrows) more frequently as compared to cells under a normal density (left panel). Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58208/58208fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58208/58208fig2.jpg" /> 
Figure 2: Comparison of cells cultured directly after isolation and after cryopreservation. The cells were cultured at a density of 1 x 105 cells per well. The gene expression of several marker transcripts was evaluated and revealed no difference between cultured cells with or without an initial cryopreservation. The boxplot shows the median of n = 3. Two-tailed Student&#39;s t-test, the p-values are included. This figure is reproduced from Baufeld and Vanselow16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58208/58208fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58208/58208fig3.jpg" /> 
Figure 3: Steroid hormone concentration in granulosa cells cultured at different plating densities. The estradiol (E2) concentration decreased significantly when GC were cultured at a high cell density, whereas the progesterone (P4) concentration only tended to be higher. The hormone concentration was corrected for the DNA content to normalize for different cell numbers. The mean and SEM of n = 3 are shown. p &#62; 0.05, two-tailed Student&#39;s t-test. This figure is reproduced from Baufeld et al.15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58208/58208fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58208/58208fig4.jpg" /> 
Figure 4: Abundance of functional key transcripts in granulosa cells cultured at a normal vs. a high plating density. Bovine GC cultured under serum-free conditions for 8 d revealed a specific regulation of several selected marker genes. The boxplot displays the median of n = 3 individual replicates. p &#60; 0.05, two-tailed Student&#39;s t-test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58208/58208fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells at JoVE.com

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

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Nanyang Technological University

Research

JoVE Journal

Biology

9.1K Views.  Leibniz Institute for Farm Animal Biology (FBN). Een lange termijn model van de cultuur van boviene granulosa cellen onder voorwaarden serumvrij wordt beschreven. Met dit model kunnen onderzoekers aan het bestuderen van de effecten van verschillende factoren en voorwaarden als verschillende plating dichtheden over de kenmerken van het oestrogeen-producerende boviene granulosa cellen.

Video: A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Medicine

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

Developmental Biology

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Bioengineering

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte&#39;s differentiation.

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. &#160;

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that ...

Establishment of a 2D Monolayer from 3D Bovine Ileal Enteroid Culture

Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating drug efficacy and toxicity, and tissue bioengineering. However, applying these models for the described reasons may be limited because of the three-dimensional (3D) nature of these models. For example, using 3D enteroid culture systems to study digestive diseases is challenging due to the inaccessibility of the intestinal lumen and its secreted substances. Indeed, stimulation of 3D organoids with pathogens requires either luminal microinjection, mechanical disruption of the 3D structure, or generation of apical-out enteroids. Moreover, these organoids cannot be co-cultured with immune and stromal cells, limiting in-depth mechanistic analysis into pathophysiological dynamics. To circumvent this, we optimized a bovine primary cell two-dimensional (2D) enteroid-derived monolayer culture system, allowing co-culture with other relevant cell types. Ileal crypts isolated from healthy adult cattle were cultured to generate 3D organoids that were cryopreserved for future use. A 2D monolayer was created using revived 3D enteroids that were passaged and disrupted to yield single cells, which were seeded on basement membrane extract-coated transwell cell culture inserts, thereby exposing their apical surface. The intestinal monolayer polarity, cellular differentiation, and barrier function were characterized using immunofluorescence microscopy and measuring transepithelial electrical resistance. Stimulation of the apical surface of the monolayer revealed the expected functionality of the monolayer, as demonstrated by cytokine secretion from both apical and basal compartments. The described 2D enteroid-derived monolayer model holds great promise in investigating host-pathogen interactions and intestinal physiology, drug development, and regenerative medicine.

Author Spotlight: Advanced Enteroid Model for Studying Host-Pathogen Interactions 

Enteroids are emerging as a novel model for studying tissue physiology and pathophysiology, drug development, and regenerative medicine. Here, we describe a bovine primary cell 2D enteroid-derived culture system that permits co-culture with relevant tissue cell types. This model offers a translational advantage for gastrointestinal research modeling.

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating ...

Immunology and Infection

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...

Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and endangered species. Research has been mostly limited to antral and large preantral follicles due to difficulty in the isolation of smaller preantral follicles, especially in large mammals such as bovine species. This work presents an efficient approach to retrieve large numbers of small preantral follicles from a single bovine ovary. The cortex of individual bovine ovaries was sliced into 500 &#181;m cubes using a tissue chopper and homogenized for 6 min at 9,000-11,000 rpm using a 10 mm probe. Large debris was separated from the homogenate using a cheese cloth, followed by serial filtration through 300 &#181;m and 40 &#181;m cell strainers. The contents retained in the 40 &#181;m strainer were rinsed into a search dish, where follicles were identified and collected into a drop of medium. The viability of the collected follicles was tested via trypan blue staining. This method enables the isolation of a large number of viable small preantral follicles from a single bovine ovary in approximately 90 min. Importantly, this method is entirely mechanical and avoids the use of enzymes to dissociate the tissue, which may damage the follicles. The follicles obtained using this protocol can be used for downstream applications such as isolation of RNA for RT-qPCR, immunolocalization of specific proteins, and in vitro culture.

Advancing the study of preantral folliculogenesis requires efficient methods of follicle isolation from single ovaries. Presented here is a streamlined, mechanical protocol for follicle isolation from bovine ovaries using a tissue chopper and homogenizer. This method allows collection of a large number of viable preantral follicles from a single ovary.

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

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