Work was funded by MRC grant G1002118.

All animal work was performed in accordance with institutional guidelines under license by the UK Home Office (project license number PPL 60/1726 and 60/4026).
Note: House animals in accordance with UK legal requirements in a 14 hr light and 10 hr dark photoperiod. Conduct experiments on animals using wild type C57Bl6J mice, Tau-GFP mice that have ubiquitous expression of green fluorescent protein (GFP)8, and Thy1-YFP mice with occasional expression of yellow fluorescent protein (YFP) in a subset of neuronal cells9: both transgenic lines were bred on a C57Bl6J background.
1. Working Conditions and Preparation of Instruments
<ol>
	<li>Perform all media preparation, tissue dissections, and culture work in a laminar flow hood to ensure sterility: this avoids the requirement of the addition of antibiotics to media.</li>
	<li>Always allow media/culture plates/embryo dishes to equilibrate for at least 1 hr, in a 37 &#176;C oven (dissection medium/embryo dishes) or 37 &#176;C, 5% CO2 incubator (culture medium and plates), prior to use.</li>
	<li>Soak glass pipettes in 0.1% solution of bovine serum albumin (BSA) for around an hour, and then leave to dry. Pull pipettes in the flame of a Bunsen burner, bending the glass as you do so, to produce finely drawn curved glass pipettes. After the pipette is pulled, make a clean cut with a glass cutter. Oven sterilize at 160 &#176;C for around 45 min. 
	NOTE: A store of these glass pipettes will be needed to transfer ovaries and follicles.</li>
</ol>
2. Preparation of Dissection and Culture Media
<ol>
	<li>Prepare Dissection Medium.
	<ol>
		<li>Dissolve 3 mg/ml BSA in Leibowitz L15 medium and filter sterilize through a 0.2 &#181;m pore, 25 mm diameter filter.</li>
	</ol>
	</li>
	<li>Neonatal Ovary Culture.
	<ol>
		<li>To prepare medium for neonatal ovary culture, make 1 ml of medium for each ovary that will be cultured. Dissolve 3 mg/ml BSA in &#945;-Minimal Essential Media (&#945;MEM) and filter sterilize through a 0.2 &#181;m pore, 13 mm diameter filter into a sterile tube.</li>
		<li>To prepare plates for neonatal ovary culture, add 1 ml of neonatal ovary culture medium into each well (one ovary per well) of a 24-well plate. Using sterile watchmaker forceps, place one polycarbonate membrane on top of the medium in each well, shiny surface up. UV sterilize membranes before use.</li>
	</ol>
	</li>
	<li>Follicle Culture.
	<ol>
		<li>To prepare medium for follicle culture, supplement &#945;-Minimal Essential Media with 1 IU/ml recombinant human follicle stimulating hormone, 5 &#956;g/ml ascorbic acid and 5% v/v serum obtained from adult female mice. Filter sterilize through a 0.2 &#181;m pore, 13 mm diameter filter into a sterile tube.</li>
		<li>Filter sterilize silicon oil through a 0.45 &#181;m pore, 25 mm diameter filter, into a sterile tube.</li>
		<li>To prepare plates, place 30 &#956;l droplets of follicle culture medium into each well of a non-tissue culture treated 96-well microtiter round-well plate, using only the top row of each plate (to allow follicles to be moved into new rows each day over the culture period). Carefully overlay medium with 70 &#956;l of sterilized silicon oil, to prevent medium evaporation.</li>
	</ol>
	</li>
	<li>Follicle-ovary Co-culture.
	<ol>
		<li>Prepare medium for follicle-ovary co-culture as for follicle culture in step 2.3.1 above, making up 1 ml of medium for each follicle-ovary co-culture being set up.</li>
		<li>To prepare plates, add 1 ml of medium into each well of a 24-well plate. Using sterile watchmaker forceps, place one Nucleopore membrane on top of the medium in each well, shiny surface up. UV sterilise membranes before use.</li>
	</ol>
	</li>
</ol>
3. Neonatal Ovary Dissection and Culture
<ol>
	<li>Place 1 ml of dissection medium into each sterile glass embryo dish.</li>
	<li>Cull neonatal mouse pups aged between postnatal day 0 and 5, culling by decapitation according to UK Home Office regulations.</li>
	<li>Grasp the skin covering the abdominal wall using a pair of fine dissection forceps and make a large incision in the skin and body wall. Pull open the incision so the entire abdomen is exposed. The bladder is usually engorged at this stage and can be punctured to make dissection easier.</li>
	<li>Move the guts out of the way using watchmaker forceps. Follow the uterine horns from the bladder up to the kidney on each side. The ovary is located just below the kidney at the top of the uterus and will appear as a cloud-like structure under a dissecting microscope.</li>
	<li>Grasp the ovary gently with watchmaker forceps, and use scissors to sever its attachment to the uterus. Transfer the pair of ovaries into embryo dishes containing pre-warmed dissection medium.</li>
	<li>Carry out fine dissection of ovaries under a dissection microscope on a heated stage (37 &#176;C). Use insulin needles to trim away the bursal sac and any excess material including the fallopian tube, until only the ovary remains.</li>
	<li>Transfer each ovary into the well of a culture plate prepared in step 2.2.2 above, using a finely drawn curved glass pipette, one ovary on top of each membrane (see Figure 1A). Culture in a 37 &#176;C, 5% CO2 incubator.</li>
	<li>Change the medium every second day. Use a pipette to exchange 50% of the medium in each well for pre-gassed fresh medium: place the pipette tip at the edge of the well of medium to avoid disturbing the membrane.</li>
	<li>Maintain cultures for up to 6 days.</li>
	<li>At the end of the culture, freeze medium and fix or freeze ovaries after a brief wash in PBS, as required.
	<ol>
		<li>Fix ovaries in Bouin&#8217;s fixative for 90 min for histological analysis, or in 10% neutral buffered formalin for 90 min for immunohistological analysis. Snap freeze ovaries by placing tissue in a polypropylene tube, placing the tube in dry ice for 5-10 min before freezing at -70 &#176;C for subsequent protein or mRNA extraction.</li>
	</ol>
	</li>
</ol>
4. Follicle Dissection and Culture
<ol>
	<li>Cull 19-23 day old female mice and isolate the ovaries. Wet the fur with 70% ethanol before making any incisions. Pinch the skin using blunt ended forceps and make a large incision in the abdomen with dissection scissors, penetrating through both the skin and the body wall.</li>
	<li>Move the gut out of the way using a finer set of dissection instruments and locate the uterus. Follow the uterus up to the ovary which is located below the kidney on each side.</li>
	<li>Gently grasping the ovary using a pair of watchmaker forceps, sever the ovaries attachment to the uterus with fine dissection scissors. Avoid collecting too much of the ovarian fat pad. Collect the ovaries and transfer into an embryo dishes containing pre-warmed dissection medium.</li>
	<li>Remove the ovarian bursa using insulin needles to trim away the bursal sac and any excess material including the fallopian tube, until only the ovary remains. Roughly halve each ovary using insulin needles.</li>
	<li>Carefully transfer each ovary-half into an individual watch glass containing 1 ml dissection medium. Cover each watch glass with a glass slide to prevent evaporation of medium and to ensure sterility.</li>
	<li>Store watch glasses containing ovary halves in a 37 &#176;C oven until needed, but carry out the next dissection step as soon as possible. Discard tissue stored in this way for more than an hour.</li>
	<li>Transfer watch glass containing an ovary half to a 37 &#176;C heated microscope stage in a laminar flow hood. Roughly dissect ovary into large pieces using two insulin needles, in order to identify late pre-antral follicles as follows: these will contain 2-3 layers of granulosa cells and have a diameter of around 180-200 &#181;m.</li>
	<li>Manually dissect out any identified follicles using one insulin needle and one 30 x 0.25 mm acupuncture needle that has been secured in a needle holder. Be careful to remove most of the surrounding stroma, but avoid damaging the basal lamina of follicles (see Figure 1B).</li>
	<li>Use a finely drawn curved glass pipette to carefully transfer dissected follicles into a collecting watch glass containing pre-warmed dissection medium. Be careful to keep follicles within the thin caliber section of pipette to avoid losing follicles.</li>
	<li>Measure follicles accurately using a calibrated eyepiece graticule fitted into a dissecting microscope.</li>
	<li>Select follicles for culture only if they measure 190 &#177; 10 &#181;m in diameter. Further select only healthy, spherical follicles for culture; these will be translucent, without dark atretic areas, and have an intact basal lamina, along with some attached thecal tissue: discard any follicles that do not fit this description. A yield of between 10-15 follicles per ovary is good.</li>
	<li>Use a finely drawn curved glass pipette to transfer a single follicle into the well of a plate made up as in step 2.3.3 above. Carefully place the follicle in the bottom of the well (and not in the upper oil layer). Culture in a 37 &#176;C, 5% CO2 incubator.</li>
	<li>Culture follicles for up to 6 days, moving follicles into a well containing fresh medium every day.
	<ol>
		<li>Prepare the next row in the 96-well plate as in plate preparation in step 2.3.3 above. Return plate to the incubator for at least an hour, to allow equilibration. Transfer follicles into fresh wells of medium, using a finely drawn glass pipette.</li>
		<li>If at any point in the culture follicles are to be left for two days before moving into fresh medium, place each follicle in a minimum of 60 &#956;l (rather than 30 &#956;l) droplets of follicle culture medium.</li>
	</ol>
	</li>
	<li>To collect data on follicle growth, measure follicles daily, using an eyepiece graticule fitted into a dissecting microscope. The oil layer will distort measurements of follicle diameter, so work out the calibration coefficient for the set-up.</li>
	<li>At the end of the culture, freeze medium and fix or freeze tissue for subsequent analysis, as in step 3.10 above.</li>
	<li>Follicle-follicle Co-culture.
	<ol>
		<li>Culture two follicles together, to investigate interactions between follicles. Culture as above, but place two follicles side-by-side, in contact, in a well.</li>
		<li>Place 100 &#956;l droplets of follicle culture medium into each well of a non-tissue culture treated 96-well microtiter flat-well plate, overlaying medium with 100 &#956;l of sterilized silicon oil. Do not transfer follicles into fresh wells as in step 4.13 above, but instead use a fine gel tip to change 50% of the medium every other day.</li>
		<li>In order to identify tissue origins within the co-culture, co-culture follicles each from a different genetic source, for example one from the ovary of a wild type mouse, and the other from the ovary of a mouse with ubiquitous expression of GFP. If using GFP or YFP tissue, minimize exposure of tissue to light as much as possible, during culture, and through the fixation/processing steps thereafter. 
		NOTE: It is normal for the two follicles to grow together into a single, &#8216;two-follicle&#8217; unit (see Figure 1C).</li>
	</ol>
	</li>
</ol>
5. Follicle-Ovary Co-Cultures
<ol>
	<li>Dissect ovaries and follicles out as in Sections 3 and 4 above.</li>
	<li>Place one neonatal ovary on top of a membrane, in a plate prepared as in step 2.4.2 above. Carefully place a single follicle in contact with one pole of the neonatal ovary, using a finely drawn curved glass pipette. Culture in a 37 &#176;C, 5% CO2 incubator for up to 5 days.</li>
	<li>In order to distinguish between the tissue origin within the co-culture, use ovaries and follicles from two different sources, for example one from a wild type mouse, and one from a mouse with ubiquitous expression of GFP.</li>
	<li>Replace 500 &#956;l of medium daily as in step 3.8 above, but using follicle culture medium. During co-culture, the follicle often becomes encapsulated by the ovary (Figure 1D).</li>
	<li>At the end of the culture, freeze medium and fix or freeze tissue for subsequent analysis, as in step 3.10 above.</li>
</ol>
6. Fixation, Immunocytochemistry and Imaging of Cultured Tissue
<ol>
	<li>At the end of culture, wash tissue in PBS and transfer to 100 &#956;l (follicle) or 1 ml (ovary) of 10% neutral buffered formalin, and fix for 1 hr on ice. Move through 3 washes of 1x PBS at 4 &#176;C.</li>
	<li>For immunocytochemistry on follicles, transfer between wells of a 96-well microtiter round-well plate using a finely drawn curved glass pipette, with each well containing different washes or treatments.</li>
	<li>For immunocytochemistry on ovaries (or ovary-follicle co-cultures), embed in 4% agarose gel and section at 50 &#181;m using a vibrotome. Float sections in the wells of a 24-well plate to apply washes or treatments, removing with a pipette.</li>
	<li>For imaging: transfer agarose sections to flat glass slides and mount with mounting medium; or transfer follicles (or follicle-follicle complexes) to cavity glass slides and mount with non-hardening mounting medium. Image specimens using a confocal microscope.</li>
</ol>

<ol>
	<li>Brien, M. J., Pendola, F. L., Eppig, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+protocol+for+in+vitro+development+of+mouse+oocytes+from+primordial+follicles+dramatically+improves+their+developmental+competence.">A revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence.</a> Biol. Reprod. 68 (5), 1682-1686 (2003).</li><li>Fortune, J. E., Yang, M. Y., Muruvi, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+earliest+stages+of+follicle+development:+follicle+formation+and+activation.">The earliest stages of follicle development: follicle formation and activation.</a> Soc. Reprod. Fertil. Suppl. 67, 203-216 (2010).</li><li>McLaughlin, M., Kinnell, H. L., Anderson, R. A., Telfer, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+phosphatase+and+tensin+homologue+(PTEN)+in+human+ovary+in+vitro+results+in+increased+activation+of+primordial+follicles+but+compromises+development+of+growing+follicles.">Inhibition of phosphatase and tensin homologue (PTEN) in human ovary in vitro results in increased activation of primordial follicles but compromises development of growing follicles.</a> Mol. Hum. Reprod. 20 (8), 736-744 (2014).</li><li>Morgan, S., Lopes, F., Gourley, C., Anderson, R. A., Spears, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cisplatin+and+doxorubicin+induce+distinct+mechanisms+of+ovarian+follicle+loss;+imatinib+provides+selective+protection+only+against+cisplatin.">Cisplatin and doxorubicin induce distinct mechanisms of ovarian follicle loss; imatinib provides selective protection only against cisplatin.</a> PLoS One. 8 (7), e70117 (2013).</li><li>Murray, A. 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=Follicular+growth+and+oocyte+competence+in+the+in+vitro+cultured+mouse+follicle:+effects+of+gonadotrophins+and+steroids.">Follicular growth and oocyte competence in the in vitro cultured mouse follicle: effects of gonadotrophins and steroids.</a> Mol Hum Reprod. 14 (2), 75-83 (2008).</li><li>Baker, S. J., Srsen, V., Lapping, R., Spears, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+effect+of+follicle-follicle+interactions+and+declining+follicle-stimulating+hormone+on+murine+follicle+health+in+vitro.">Combined effect of follicle-follicle interactions and declining follicle-stimulating hormone on murine follicle health in vitro.</a> Biol. Reprod. 65 (4), 1304-1310 (2001).</li><li>Campbell, L., Trendell, J., Spears, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cells+migrating+from+the+thecal+layer+of+ovarian+follicles.">Identification of cells migrating from the thecal layer of ovarian follicles.</a> Cell Tissue Res. 353 (1), 189-194 (2013).</li><li>Pratt, T., Sharp, L., Nichols, J., Price, D. J., Mason, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+and+transgenic+mice+ubiquitously+expressing+a+tau-tagged+green+fluorescent+protein.">Embryonic stem cells and transgenic mice ubiquitously expressing a tau-tagged green fluorescent protein.</a> Dev. Biol. 228 (1), 19-28 (2000).</li><li>Feng, G., 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=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28 (1), 41-51 (2000).</li><li>Maiani, E., 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=Reply+to:+cisplatin-induced+primordial+follicle+oocyte+killing+and+loss+of+fertility+are+not+prevented+by+imatinib.">Reply to: cisplatin-induced primordial follicle oocyte killing and loss of fertility are not prevented by imatinib.</a> Nat. Med. 18 (8), 1172-1174 (2012).</li><li>Spears, N., Bruin, d. r., Gosden, J. P., G, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+establishment+of+follicular+dominance+in+co-cultured+mouse+ovarian+follicles.">The establishment of follicular dominance in co-cultured mouse ovarian follicles.</a> J. Reprod. Fertil. 106 (1), 1-6 (1996).</li><li>Desmeules, P., Devine, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+ovotoxicity+of+cyclophosphamide+metabolites+on+cultured+mouse+ovaries.">Characterizing the ovotoxicity of cyclophosphamide metabolites on cultured mouse ovaries.</a> Toxicol. Sc. 90 (2), 500-509 (2006).</li><li>Tagler, D., 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=Promoting+extracellular+matrix+remodelling+via+ascorbic+acid+enhances+the+survival+of+primary+ovarian+follicles+encapsulated+in+alginate+hydrogels.">Promoting extracellular matrix remodelling via ascorbic acid enhances the survival of primary ovarian follicles encapsulated in alginate hydrogels.</a> Biotechnol. Bioeng. 111 (7), 1417-1429 (2014).</li></ol>

The authors have nothing to disclose.

We describe here various culture systems that can be utilized to support the development of mouse ovarian follicles in vitro, using whole neonatal ovaries containing only the earliest follicle stages, individual preantral ovarian follicles and also co-cultures of the two tissues.
Culture of neonatal mouse ovaries supports early ovarian development, particularly follicle growth initiation up to the secondary follicle stage. A range of ages of neonatal mice can be used for these cultures, depending on the developmental stages of interest. If ovaries are obtained from newborn mice, follicle formation will be underway but not yet complete: culture will at least partially support continued follicle formation followed by follicle growth. Alternatively, use of ovaries from mice around four-to-five days of age (by which time follicle formation is already complete) results in culture of a greater number of primordial and growing follicles12. Beneficial aspects of the specific technique described here include use of floating polycarbonate membranes, which allow greater oxygenation of the tissue, and culture in a very basic medium consisting of only &#945;MEM and BSA, avoiding the use of undefined additives such as serum. Culture of whole ovaries does not seem to support development beyond the secondary follicle stage, with subsequent development requiring a change in technique, such as the dissection of follicle-granulosa cell complexes from the cultured neonatal ovary1.
Later stages of follicle development can be developed in vitro by dissecting out individual, intact late pre-antral follicles, which can be grown to the preovulatory stage in culture whilst maintaining their three-dimensional structure. The use of intact follicles for this culture technique maintains the relationship between the different follicular components as occurs in vivo. This culture system can be used to obtain oocytes that can support fertilisation and subsequent embryo development.
Fertilizable oocytes can also be obtained from mouse neonatal ovaries using an initial culture protocol much as described here, followed by a second stage during which oocyte-granulosa cell complexes are grown in vitro1. Other systems used fairly frequently today include culture of follicles or ovarian tissue that has been encapsulated in a material such as alginate hydrogel, to provide support (see, for example, Tagler et al.13). Much of the focus of method development now is to improve culture techniques for the ovaries and follicles of larger mammals, with the long-term aim of obtaining fertilisable oocytes from primordial follicles from a range of species, including humans.
At any one time, mammalian ovaries contain follicles at a range of developmental stages, with interactions between follicles affecting their regulation. This aspect of ovarian function is poorly understood and difficult to examine in vivo. The final method described here uses co-culture systems to support development of different stages of follicles in vitro. If required, one or both tissues can be pre-treated in vivo or in vitro prior to co-culture. Co-culture systems such as this provide an ideal way in which to examine follicle-follicle interactions, for instance how growing, antral follicles affect the primordial follicle pool, aspects of ovarian biology that have proven difficult to examine until now.
Whole ovary culture techniques are fairly straightforward, although careful dissection is required to avoid accidental tissue damage. Dissection of individual follicles is a specialised technique, requiring repeated practice before follicles at the right stage can be dissected out from the ovary intact and undamaged. It is critical to dissect out individual follicles carefully, or damage sustained during the dissection protocol can result in follicle death during the subsequent culture period. Where follicles are placed directly into the well of microtiter plates, it is important to use only non-tissue culture treated plastics, to minimise plating down of thecal cells on the plastics: if tissue culture treated plasticware is used, the follicles will attach to the base of the well and rupture as they grow. For all co-culture work, tissues must be placed directly in contact with each other.
The medium detailed above for use in the follicle culture technique includes the addition of mouse serum. It is possible to replace the mouse serum with fetal bovine serum, but only occasional batches of such sera will fully support follicle development to the preovulatory stage, with batch-testing required to identify suitable sources. Batch-testing of FSH is also advisable, as the International Units by which FSH is assessed correlate only crudely to follicle growth in vitro. If follicle rupture routinely occurs during the culture period, consider replacing stock ascorbic acid with a fresh batch.
The techniques do not require particularly specialised equipment other than dissecting microscopes and tissue culture incubators, although use of a laminar flow hood and good sterile technique allow the ovarian follicles to be cultured in the absence of antibiotics, as in the methods described here. This can be helpful, to avoid any potential detrimental effect of antibiotics on the oocytes, particularly if they are to be fertilized following culture. Where it is not possible to work in a sterile environment, it is advisable to add antibiotics to the dissection and culture media.

The mammalian ovary is composed of a female&#8217;s lifetime supply of oocytes, each contained within an ovarian follicle. The follicles are formed prior to birth at the resting, primordial stage: once the follicle pool is established, there is a continual and gradual movement of follicles from the primordial into the growing follicle pool. As follicles start to grow, they develop through the primary, secondary, preantral and then antral stages, until they reach the preovulatory, or Graafian, stage. Only oocytes from preovulatory follicles have full developmental competence, able to support embryonic development to term, if fertilized.
The ovary has long been known to develop in a highly physiological manner in vitro. This is likely to be due to each ovarian follicle containing within it the cells necessary to support an oocyte from the immature stage (at which point it is unable to complete its meiotic division) through to the developmentally competent stage (at which point it can fully support the completion of meiosis, fertilization and the development of the resulting embryo to term).
The physiological nature of ovary development in vitro has led to the wide use of ovary culture techniques. Consequently, in vitro methods have been used to investigate the regulation of ovary development, ovarian pathology (for instance that of polycystic ovarian syndrome, PCOS), examination of how ovaries/oocytes are affected by exposure to chemicals, and also with the practical aim of obtaining fertilizable oocytes from primordial ovarian follicles. To date, the latter has been achieved only using mouse ovarian tissue1, although culture techniques using follicles from large mammals, including humans, have greatly improved in recent years2,3.
We describe here several culture methods using mouse ovarian tissue. The first method uses whole neonatal mouse ovaries, and supports the formation and early development of primordial follicles4. The second system supports the growth of individual, intact ovarian follicles from the late preantral to the preovulatory stage; using this technique, follicles can be cultured individually, or in pairs5,6. Finally, we describe a co-culture system, combining the first two techniques in a method that allows investigation of the interactions between growing and primordial ovarian follicles7.
Culture of individual intact ovarian follicles allows follicle growth to be determined from daily follicle measurements during the culture period, while medium analysis allows investigation of follicle/ovary hormone production. Further tissue analyses can be achieved by a collection of follicles or ovarian tissue at the end of culture, for histological/immunohistological analyses or for subsequent processing for example to obtain mRNA or proteins.

<table border="0" cellpadding="0" cellspacing="0" width="740">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Name of the Material/Equipment</td>
			<td style="width:159px;">Company</td>
			<td style="width:151px;">Catalog Number</td>
			<td style="width:265px;">Comments/Description</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Leibowitz L15</td>
			<td style="width:159px;">Invitrogen</td>
			<td style="width:151px;">11415049</td>
			<td style="width:265px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">&#945;MEM</td>
			<td style="width:159px;">Invitrogen</td>
			<td style="width:151px;">22571020</td>
			<td style="width:265px;">Supplied as sodium bicarbonate buffered (2200 mg/L)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Bovine serum albumin</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:151px;">A9418</td>
			<td style="width:265px;">For dissection medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Bovine serum albumin</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:151px;">A3311</td>
			<td style="width:265px;">For culture medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Bovine serum albumin</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:151px;">A2153</td>
			<td style="width:265px;">For coating glass pipettes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Mouse serum</td>
			<td style="width:159px;">-</td>
			<td style="width:151px;">-</td>
			<td style="width:265px;">Collected from cardiac puncture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">FSH</td>
			<td style="width:159px;">Merck Serono</td>
			<td style="width:151px;">Gonal F</td>
			<td style="width:265px;">Batch testing is often necessary. Make up stock solution of 1IU/10&#181;l in &#945;MEM and store at -20&#176;C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Ascorbic acid</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:151px;">A4034</td>
			<td style="width:265px;">Make up stock of 5mg/ml in &#945;MEM and store at -20&#176;C.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Silicon oil</td>
			<td style="width:159px;">VWR</td>
			<td style="width:151px;">630064V</td>
			<td style="width:265px;">Dow Corning Silicone Fluid 200/50cS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Syringe filters (25mm, 0.2&#181;m)</td>
			<td style="width:159px;">Greiner</td>
			<td style="width:151px;">16532K</td>
			<td style="width:265px;">Cellulose acetate filter: for filtering larger (&#62; 5mls) volumes of medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Syringe filters (13mm, 0.2&#181;m)</td>
			<td style="width:159px;">Iwaki</td>
			<td style="width:151px;">3032-013</td>
			<td style="width:265px;">Cellulose acetate filter: for filtering smaller (&#60; 5mls) volumes of medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Syringe filters (25mm, 0.45&#181;m)</td>
			<td style="width:159px;">Iwaki</td>
			<td style="width:151px;">2053-025</td>
			<td style="width:265px;">Cellulose acetate filter: for filtering oil.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Sterile tubes</td>
			<td style="width:159px;">Greiner</td>
			<td style="width:151px;">187261</td>
			<td style="width:265px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">24 well plate</td>
			<td style="width:159px;">Greiner</td>
			<td style="width:151px;">662160</td>
			<td style="width:265px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">96 well round bottom plate</td>
			<td style="width:159px;">Iwaki</td>
			<td style="width:151px;">3875-096</td>
			<td style="width:265px;">Use only non-tissue culture treated plates</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">96 well flat bottomed well</td>
			<td style="width:159px;">Iwaki</td>
			<td style="width:151px;">3860-096</td>
			<td style="width:265px;">Use only non-tissue culture treated plates</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Whatman nucleopore membranes</td>
			<td style="width:159px;">Camlab</td>
			<td style="width:151px;">WN/110414</td>
			<td style="width:265px;">Use shiny surface up, polycarbonate, 13mm diameter, 8.0&#181;m pore size</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Insulin needles</td>
			<td style="width:159px;">BD medical supplies</td>
			<td style="width:151px;">037-7606</td>
			<td style="width:265px;">0.33mm (29G) +1ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Glass embryo dishes</td>
			<td style="width:159px;">VWR</td>
			<td style="width:151px;">720-0579</td>
			<td style="width:265px;">Sterilise then warm before use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Glass pipettes</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:151px;">10209381</td>
			<td style="width:265px;">BSA coated before use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Gel tips</td>
			<td style="width:159px;">AlphaLabs</td>
			<td style="width:151px;">LW1103</td>
			<td style="width:265px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Acupuncture needles</td>
			<td style="width:159px;">Acumedic LTD</td>
			<td style="width:151px;">30mmx0.25 Type C</td>
			<td style="width:265px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bouin&#8217;s fixative&#160;</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:151px;">HT10132-1L</td>
			<td style="width:265px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td>HT5014-120ml</td>
			<td style="width:265px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">1.5 ml polypropylene tubes</td>
			<td style="width:159px;">Greiner BioOne</td>
			<td align="right">616201</td>
			<td style="width:265px;"></td>
		</tr>
	</tbody>
</table>

culture and co-culture of mouse ovaries and ovarian follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

Figure 1 shows images of ovaries and follicles at the start of, and during, the culture procedures.
Figure 2 shows representative results of neonatal ovaries (here, ovaries from newborn pups) exposed to reproductive toxicants during culture. Neonatal ovaries were cultured for 6 days, and exposed to a chemotherapy drug, either cisplatin or doxorubicin, on Day 2 of culture. At the end of the culture, ovaries were fixed, processed for histology, and ovaries then examined to determine the number of healthy and unhealthy ovarian follicles4. Alternatively, a more rapid assessment of early growing follicles can be obtained from the culture of ovarian fragments, using ovaries from a mouse expressing an oocyte-specific fluorescent marker10.
During the culture of individual follicles, follicle growth can be easily ascertained by daily measurements during the culture period. Figure 3 shows representative results of the growth of follicles cultured in the presence or absence of the gonadotrophin hormones follicle stimulating hormone (FSH) and luteinizing hormone (LH)5.
Follicle-follicle or follicle-ovary co-culture can be used to investigate interactions between follicles. Depending on the tissue and processing, images can be obtained using bright-field, fluorescence or confocal microscopes. Figure 4 shows representative results of images from such co-cultures, examining various cell types involved in follicle-follicle communication, including endothelial and neuronal cells7.
<img alt="Figure 1" src="/files/ftp_upload/52458/52458fig1highres.jpg" width="650" /> 
Figure 1: (A) Photomicrograph of a neonatal ovary obtained from a mouse on the day of birth, placed on a polycarbonate membrane. (B) Photomicrograph of freshly dissected healthy ovarian follicle, with most of the surrounding stromal tissue dissected away. (C) Photomicrographs of co-culture of two ovarian follicles over the first three days of culture, showing the close contact that develops between the two follicles as the culture proceeds (Ci: first day of culture; Cii: second day of culture; Ciii: third day of culture). Reproduced from Spears et al.11 (D) Photomicrograph of co-cultured preantral follicle and neonatal ovary after two days of culture. Image shows the follicle becoming encapsulated by the ovary. Arrow shows preantral follicle. Image from Dr Federica Lopes. <a href="https://www.jove.com/files/ftp_upload/52458/52458fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52458/52458fig2.jpg" /> 
Figure 2: Effect of the chemotherapy drugs cisplatin and doxorubicin on neonatal cultured ovaries. Cisplatin and doxorubicin both lead to loss of follicle health and a reduction in follicle numbers. (A) Cisplatin; (B) Doxorubicin: (i) Percentage of unhealthy follicles (clear); and (ii) total number of follicles (shaded) in each ovary. Bars denote mean + sem; n = 5 for all groups, stars denote significant differences relative to control (*p &#60;0.05, **p &#60;0.01, ***p &#60;0.001). Reproduced from Morgan et al.4
<img alt="Figure 3" src="/files/ftp_upload/52458/52458fig3.jpg" width="400" /> 
Figure 3: Growth rates of follicles exposed to different gonadotrophin environments. Values are mean &#177; SEM (n &#8805;16 for each group). Arrow indicates the start of antral formation in all gonadotrophin groups. Reproduced from Murray et al.5
<img alt="Figure 4" src="/files/ftp_upload/52458/52458fig4highres.jpg" width="650" /> 
Figure 4: Images from follicle-follicle and ovary-follicle co-cultures, showing follicle-follicle interactions. (A) Confocal micrograph of a green fluorescent protein (GFP) follicle and a wild-type (WT) follicle after co-culture (GFP expression shown here in white), with processes from the GFP follicle shown extending over the WT follicle. (B) Cross-section through a GFP/WT follicle complex following co-culture, showing that GFP processes (green) from the adjoining follicle lie adjacent to the basal lamina of the WT follicle. (C) Neonatal WT ovary following culture with a pre-antral GFP-expressing follicle, showing that GFP cells and cellular processes (green) have migrated through the ovarian interstitum of the neonatal ovary. (D) GFP/WT follicle complex containing endothelial cells, shown by expression of endothelial cell marker CD31 (red). (E) GFP/WT follicle complex containing neuronal cells, shown by expression of neuronal marker beta tubulin III (see here as red, or as yellow if double-labelled with GFP, green). (F) YFP/WT follicle complex, where YFP refers to follicle from a Thy1-YFP mouse that has occasional expression of yellow fluorescent protein (YFP: yellow) in a subset of neuronal cells9, showing that neurons extend from the YFP follicle. Bars 50&#160;&#956;m (A, B, D), 100&#160;&#956;m (C, E, F), 100&#160;&#956;m (inset in A), 10&#160;&#956;m (inset in C), 15&#160;&#956;m (inset in D). Reproduced from Campbell et al.7 <a href="https://www.jove.com/files/ftp_upload/52458/52458fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles at JoVE.com

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

culture-and-co-culture-of-mouse-ovaries-and-ovarian-follicles

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JoVE Journal

Developmental Biology

22.4K Views.  University of Edinburgh. This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

Video: Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

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, ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies have demonstrated the wide differentiation capacity that these cells possess. The ovary represents a promising candidate for cell-based therapies due to the fact that it is rich in MSCs and that it is frequently discarded after ovariectomy surgeries as biological waste. This article describes procedures for the isolation, expansion, and differentiation of MSCs derived from the canine ovary, without the necessity of cell-sorting techniques. This protocol represents an important tool for regenerative medicine because of the broad applicability of these highly differentiable cells in clinical trials and therapeutic uses.

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...