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>

<ol>
	<li>Gutierrez, C. G., Ralph, J. H., Telfer, E. E., Wilmut, I., 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=Growth+and+antrum+formation+of+bovine+preantral+follicles+in+long-term+culture+in+vitro.">Growth and antrum formation of bovine preantral follicles in long-term culture in vitro.</a> Biology of Reproduction. 62 (5), 1322-1328 (2000).</li><li>Cortvrindt, R., Hu, Y., Smitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+luteinizing+hormone+as+a+survival+and+differentiation+factor+increases+oocyte+maturation+in+recombinant+follicle+stimulating+hormone-supplemented+mouse+preantral+follicle+culture.">Recombinant luteinizing hormone as a survival and differentiation factor increases oocyte maturation in recombinant follicle stimulating hormone-supplemented mouse preantral follicle culture.</a> Human Reproduction. 13 (5), 1292-1302 (1998).</li><li>Paes, V. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+granulosa+cell+lines.">Ovarian granulosa cell lines.</a> Molecular and Cellular Endocrinology. 228 (1-2), 67-78 (2004).</li><li>Bernath, V. A., 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=Cyclic+AMP+inhibits+fibronectin+gene+expression+in+a+newly+developed+granulosa+cell+line+by+a+mechanism+that+suppresses+cAMP-responsive+element-dependent+transcriptional+activation.">Cyclic AMP inhibits fibronectin gene expression in a newly developed granulosa cell line by a mechanism that suppresses cAMP-responsive element-dependent transcriptional activation.</a> The Journal of Biological Chemistry. 265 (30), 18219-18226 (1990).</li><li>Lerner, A. A., Salamone, D. F., Chiappe, M. E., Baranao, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+studies+between+freshly+isolated+and+spontaneously+immortalized+bovine+granulosa+cells:+protein+secretion,+steroid+metabolism,+and+responsiveness+to+growth+factors.">Comparative studies between freshly isolated and spontaneously immortalized bovine granulosa cells: protein secretion, steroid metabolism, and responsiveness to growth factors.</a> Journal of Cellular Physiology. 164 (2), 395-403 (1995).</li><li>Gutierrez, C. G., Campbell, B. K., 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=Development+of+a+long-term+bovine+granulosa+cell+culture+system:+induction+and+maintenance+of+estradiol+production,+response+to+follicle-+stimulating+hormone,+and+morphological+characteristics.">Development of a long-term bovine granulosa cell culture system: induction and maintenance of estradiol production, response to follicle- stimulating hormone, and morphological characteristics.</a> Biology of Reproduction. 56 (3), 608-616 (1997).</li><li>Campbell, B. K., Scaramuzzi, R. 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. A. <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+follicle-stimulating+hormone+on+steroid+secretion+and+messenger+ribonucleic+acids+encoding+cytochromes+P450+aromatase+and+cholesterol+side-chain+cleavage+in+bovine+granulosa+cells+in+vitro.">Effect of follicle-stimulating hormone on steroid secretion and messenger ribonucleic acids encoding cytochromes P450 aromatase and cholesterol side-chain cleavage in bovine granulosa cells in vitro.</a> Biology of Reproduction. 62 (1), 186-191 (2000).</li><li>Portela, V. M., Zamberlam, G., 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=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 Ca2+, Mg2+</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 &#181;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 &#181;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>&#945;-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 &#181;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 &#181;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/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/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/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/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 ...

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

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JoVE Journal

Biology

9.0K Views.  Leibniz Institute for Farm Animal Biology (FBN). 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.

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

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique tissue important for proper function of the synovial joint and is constituted by a dense extracellular matrix (ECM), rich in proteoglycan and type II collagen. Chondrocytes are the only cell type present within cartilage and are widespread and relatively low in number. Altered external stimuli and cellular signalling can lead to changes in ECM composition and deterioration, which are important pathological hallmarks in diseases such as osteoarthritis (OA) and rheumatoid arthritis.
Ex vivo cartilage models allow 1) profiling of chondrocyte mediated alterations of cartilage tissue turnover, 2) visualizing the cartilage ECM composition, and 3) chondrocyte rearrangement directly in the tissue. Profiling these alterations in response to stimuli or treatments are of high importance in various aspects of cartilage biology, and complement in vitro experiments in isolated chondrocytes, or more complex models in live animals where experimental conditions are more difficult to control.
Cartilage explants present a translational and easily accessible method for assessing tissue remodeling in the cartilage ECM in controllable settings. Here, we describe a protocol for isolating and culturing live bovine cartilage explants. The method uses tissue from the bovine knee, which is easily accessible from the local butchery. Both explants and conditioned culture medium can be analyzed to investigate tissue turnover, ECM composition, and chondrocyte function, thus profiling ECM modulation.

Here, we present a protocol describing isolation and culturing of cartilage explants from bovine knees. This method provides an easy and accessible tool to describe tissue changes in response to biological stimuli or novel therapeutics targeting the joint.

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique ...

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of these methods to non-model organisms. A series of protocols to optimize tissue collection from bats has been developed for a series of high-throughput sequencing approaches. Outlined here are protocols for the capture of bats, desired demographics to be collected for each bat, and optimized methods to minimize stress on a bat during tissue collection. Specifically outlined are methods for collecting and treating tissue to obtain (i) DNA for high molecular weight genomic analyses, (ii) RNA for tissue-specific transcriptomes, and (iii) proteins for proteomic-level analyses. Lastly, also outlined is a method to avoid lethal sampling by creating viable primary cell cultures from wing clips. A central motivation of these methods is to maximize the amount of potential molecular and morphological data for each bat and suggest optimal ways to preserve tissues so they retain their value as new methods develop in the future. This standardization has become particularly important as initiatives to sequence chromosome-level, error-free genomes of species across the world have emerged, in which multiple scientific parties are spearheading the sequencing of different taxonomic groups. The protocols outlined herein define the ideal tissue collection and tissue preservation methods for Bat1K, the consortium that is sequencing the genomes of every species of bat.

This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue.

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological substances or characterization of specific biological activities. However, due to the lack of universally applicable cell culture media and protocols, well described cell culture methods for marine organisms are still limited. Meanwhile, the commonly occurring microbial contamination and polytropic properties of marine invertebrate cells further impede the establishment of an effective cell culture strategy for marine invertebrates. Here, we describe an easy-to-handle method for culturing intestinal cells from sea cucumber Apostichopus japonicus; additionally, we provide an example of in vitro apoptosis induction and detection in primary cultured intestinal cells. Moreover, this experiment provides details about the appropriate culture medium and cell collection method. The described protocol is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea, and it can provide sufficient cells for multiple in vitro experimental applications. This technique would enable researchers to efficiently manipulate primary cell cultures from marine invertebrates and to facilitate the functional evaluation of targeted biological materials on cells.

This protocol provides an easy-to-handle method to culture the intestinal cells from sea cucumber Apostichopus japonicus and is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea.

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological ...

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 ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...