This work was funded by the National Institutes of Health, National Institute of Child Health and Human Development (NICHD) F31 HD089693, the National Institute for Environmental Health Sciences / National Center for Advancing Translational Sciences (NIEHS/NCATS) UH3TR001207 and 4UH3ES029073-03, and the Thomas J. Watkin&#8217;s Memorial Professorship.
The authors would like to thank Eric W. Roth for their assistance with transmission electron microscopy. This work made use of the BioCryo facility of Northwestern University&#8217;s NUANCE Center, which has received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-1542205); the MRSEC program (NSF DMR-1720139) at the Materials Research Center; the International Institute for Nanotechnology (IIN); and the State of Illinois, through the IIN. It also made use of the CryoCluster equipment, which has received support from the MRI program (NSF DMR-1229693). Graphics in Figure 1 were designed using BioRender.com.

All mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Northwestern University, and all procedures were performed under IACUC-approved protocols.
1. Preparation of enzymatic tissue-dissociation solutions
<ol>
	<li>Use two different enzymatic solutions (Solution 1 and Solution 2), both made using a basal culture medium solution (BM).</li>
	<li>To prepare BM, add serum and penicillin-streptomycin to minimum essential medium to final concentrations of ...

With the completion of this organoid generation protocol, the user will have four different culture techniques available to them for assembling testicular constructs and organoids in either ECM or ECM-free environments. Importantly, all four methods allow the researcher to non-invasively observe organoid self-assembly over time through time-lapse imaging or video recording, and to noninvasively collect conditioned media for analysis of secreted hormones and cytokines, without disturbing tissues during culture. In all met...

Testicular organoids are a pioneering technique for studying testicular development, spermatogenesis, and physiology in vitro1,2,3,4. Several methods have been explored for organoid generation; these include a variety of extracellular matrix (ECM) and ECM-free culture systems, in both two-dimensional (2D) and three-dimensional (3D) orientations. Different generation methods can promote distinct cellular assembly strategies; this results in a high level of morphological and functional variability between published organoid models. The purpose of this article is to discuss the current state of in vitro testicular models, and to serve as a template for future investigators, when designing testicular organoid experiments. Within the present study, four different culture system archetypes are defined and characterized in experimental process and biological outcome. These include: 2D ECM-Free, 2D ECM, 3D ECM-Free, and 3D ECM culture methods. The strategies presented herein are intended to be simple, accessible, and highly reproducible between different laboratories and research groups.
Historically for the testis, the designation &#8220;in vitro&#8221;, has been used for several different culture methods of testicular tissues and cells. These include organotypic tissue/organ culture methods (i.e., explant culture)5, isolated seminiferous tubule culture6, testicular cell culture7, and methods of de novo tissue morphogenesis (i.e., biological constructs and organoids)1. The first investigations into in vitro spermatogenesis were performed approximately 100 years ago, with the culture of rabbit testis explants in 19208, and later in 1937 with mouse explants9. Within these initial experiments spermatogonia were observed to largely degenerate across the first week of culture, though some meiotically differentiating cells were identified. Reminiscent of these historical reports, testis explant culture was revived and optimized in 2011 to become a feasible technique for studying the testis10. Since 2011, explant culture has produced fertility competent sperm in multiple reports11,12,13. Yet, due to explant culture&#8217;s reliance upon pre-existing native testis tubules, these recent advances are more accurately described as examples of &#8220;ex vivo&#8221; testicular function and spermatogenesis, tissue function that was maintained or resumed upon removal from an organism&#8217;s body. Despite its prevalence in the literature, long-term germ cell maintenance and differentiation within testicular explants is challenging to replicate14,15,16,17,18, especially over timeframes long enough to fully observe in vitro spermatogenesis (~35 days in mice19 and 74 in humans20). It is intriguing to appreciate that many of the same challenges experienced 100 years ago, are still experienced within ex vivo spermatogenesis today.
Different than ex vivo approaches, testicular organoids are de novo assembled microtissues generated entirely in vitro from cellular sources (i.e., primary testicular cells). Testicular organoids provide a creative strategy to circumvent the field&#8217;s historical reliance upon pre-existing native tissue, and to recapitulate testicular biology completely in vitro. There are multiple requirements shared by most organoid tissue models; these include (1) in vivo-mimetic tissue morphology or architecture, (2) multiple major cell types of the represented tissue, (3) self-assembly or self-organization in their generation, and (4) the ability to simulate some level of the represented tissue&#8217;s function and physiology21,22,23,24. For the testis, this can be captured in four major hallmarks: (1) the inclusion of major testicular cell types, germ, Sertoli, Leydig, peritubular, and other interstitial cells, (2) cell-directed tissue assembly, (3) appropriately-compartmentalized cell types into separate tubular compartments (germ and Sertoli) and interstitial regions (all other cell types), and (4) some degree of tissue function (e.g., reproductive hormone secretion or tissue responses, and germ cell maintenance and differentiation). Considering the historic challenges in maintaining germ cell differentiation ex vivo and in vitro, the recapitulation of in vivo-mimetic testicular architectures (i.e., structures resembling seminiferous tubules) with additional markers suggesting simulation of testicular physiology (e.g., endocrine function), are priority milestones towards generating organoids which might one day sustain in vitro spermatogenesis.
The majority of published testicular organoid methods take advantage of commercially available ECM (e.g., collagen or proprietary ECM formulations)25,26,27 or custom-sourced ECMs (i.e., decellularized testis ECM-derived hydrogels)28,29,30. Exogenous ECM promotes de novo tissue formation through providing an assembly-supportive scaffold for tissue generation. ECM methods have afforded an impressive level of tissue formation, including some germ cell presence and tissue-mimetic morphology25,28. However, the ECMs they utilize are not always universally available (i.e., decellularized ECM-derived hydrogels), and some methods require sophisticated gel and cell seeding orientations (e.g., 3-layer gradients of ECM and 3D printing)25,31,32. Scaffold-free methods (e.g., hanging drop and nonadherent culture plates)33,34,35 have also generated robust and highly reproducible organoids without the need of ECM gels or scaffolds. However, the tissue morphology of these scaffold-free organoids is often dissimilar to in vivo testes, and most of these reports incorporate a biochemical ECM additive to promote tissue formation33,34,36, or alternatively, rely upon centrifugation for forced cell aggregation and compaction34, making them less ideal for studying cell-directed migration and self-organization.
The four organoid generation methods presented in this manuscript include both ECM-dependent and independent strategies, each using simple cell seeding that enables the observation of cell-driven organoid self-assembly. All four techniques can be performed from the same cell suspensions or can make use of custom and cell-type enriched populations. A strength of these methods is the ability to observe organoids self-assemble in real-time, and to directly compare how testicular structures self-assemble between different culture microenvironments. The phenotypic differences between these four culture methods should be considered for their impact on the research question or subject of the investigator. Each method produces biological constructs or organoids within 24 h or less. In conclusion, the methods presented here provide a toolkit of organoid assembly techniques for studying testicular organoid assembly, tissue development, and testicular physiology in vitro.

<table>
	<tbody>
		<tr>
			<td>0.22 um Media Sterile Filters</td>
			<td>Millipore Sigma</td>
			<td>scgpu05re</td>
			<td>For sterile filtering media</td>
		</tr>
		<tr>
			<td>3&#946;HSD primary antibody</td>
			<td>Cosmo Bio Co</td>
			<td>K0607</td>
			<td>Leydig cell marker, 1:500 dilution</td>
		</tr>
		<tr>
			<td>AlexaFluor 568 &#945;-Mouse</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21202</td>
			<td>Fluorescence-tagged secondary antibody</td>
		</tr>
		<tr>
			<td>AlexaFluor 568 &#945;-Rabbit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10042</td>
			<td>Fluorescence-tagged secondary antibody</td>
		</tr>
		<tr>
			<td>Alpha Minimum Essential Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11-095-080</td>
			<td>Base of culture media</td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Worthington Bio</td>
			<td>LS004197</td>
			<td>For dissociation solution 1</td>
		</tr>
		<tr>
			<td>Corning Matrigel Membrane Matrix, LDEV-free</td>
			<td>Corning</td>
			<td>354234</td>
			<td>Extracellular matrix used for casting 2D and 3D ECM culture gels</td>
		</tr>
		<tr>
			<td>Countess Cell counter</td>
			<td>Thermo Fisher Scientific</td>
			<td>C10227</td>
			<td>Autmated cell counter (hemacytometer machine)</td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>C10228</td>
			<td>Hemacytometer slide for use with Countess automated counter</td>
		</tr>
		<tr>
			<td>DDX4 primary antibody</td>
			<td>Abcam</td>
			<td>138540</td>
			<td>Spermatogonia marker, 1:500 dilution</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I (2,280 u/mgDW)</td>
			<td>Worthington Bio</td>
			<td>LS002140</td>
			<td>For dissociation solution 1</td>
		</tr>
		<tr>
			<td>DPBS 1X, + CaCl + MgCl</td>
			<td>Thermo Fisher Scientific</td>
			<td>14040182</td>
			<td>For reconstituting Hyaluronidase</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline +Ca/+Mg</td>
			<td>Thermo Fisher Scientific</td>
			<td>14040117</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Embryo Grade H2O</td>
			<td>MIllipore Sigma</td>
			<td>W1503</td>
			<td>For reconstituting Collagenase I and Dnase I</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000044</td>
			<td>For quencing enzyme dissocation solutions</td>
		</tr>
		<tr>
			<td>Follicle stimulating hormone</td>
			<td>Abcam</td>
			<td>ab51888</td>
			<td>For long-term organoid culture</td>
		</tr>
		<tr>
			<td>Human chorionic gonadotropin</td>
			<td>Millipore Sigma</td>
			<td>C1063</td>
			<td>For long-term organoid culture</td>
		</tr>
		<tr>
			<td>Hyaluronidase, from bovine testes</td>
			<td>Millipore Sigma</td>
			<td>H4272</td>
			<td>For dissociation solution 2</td>
		</tr>
		<tr>
			<td>Inhibin B Enzyme-linked Immunosorbent Assay</td>
			<td>Ansh Labs</td>
			<td>AL-107</td>
			<td>Inhibin B ELISA Kit</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Thermo Fisher Scientific</td>
			<td>10828-028</td>
			<td>Serum source for Basal media</td>
		</tr>
		<tr>
			<td>MicroTissues 3D Petri Dish micro-mold spheroids (24-35, 5x7 array)</td>
			<td>Millipore Sigma</td>
			<td>Z764051</td>
			<td>For 3D ECM-Free organoid fabrication</td>
		</tr>
		<tr>
			<td>Nunc, Lab Tek II Chamber Slide System, 4-well</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-565-7</td>
			<td>For 2D ECM-free, and 2D, 3D ECM culture</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15-140-122</td>
			<td>Antibiotic for media</td>
		</tr>
		<tr>
			<td>Richard-Allan Scientific; Histogel, Specimen processing gel</td>
			<td>Thermo Fisher Scientific</td>
			<td>HG-4000-012</td>
			<td>For aiding paraffin embedding</td>
		</tr>
		<tr>
			<td>SOX9 primary antibody</td>
			<td>Millipore Sigma</td>
			<td>AB5535</td>
			<td>Sertoli Marker, 1:500 dilution</td>
		</tr>
		<tr>
			<td>Tedklad Global Mouse Chow (Breeder)</td>
			<td>Teklad Global</td>
			<td>2920</td>
			<td>Mouse food without phytoestrogens</td>
		</tr>
		<tr>
			<td>Tedklad Global Mouse Chow (Maintenance)</td>
			<td>Teklad Global</td>
			<td>2916</td>
			<td>Mouse food without phytoestrogens</td>
		</tr>
		<tr>
			<td>Testosterone Enzyme-linked Immunosorbent Assay</td>
			<td>Calbiotech</td>
			<td>TE373S</td>
			<td>Testosterone ELISA Kit</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Thermo Fisher Scientific</td>
			<td>15250061</td>
			<td>For cell counting</td>
		</tr>
		<tr>
			<td>&#945;SMA primary antibody</td>
			<td>Millipore Sigma</td>
			<td>A2547</td>
			<td>Peritubular marker, 1:500 dilution</td>
		</tr>
		<tr>
			<td>&#946;Catenin primary antibody</td>
			<td>BD Biosciences</td>
			<td>610154</td>
			<td>Sertoli Cytoplasm marker, 1:100 dilution</td>
		</tr>
	</tbody>
</table>

extra cellular matrix-based and extra cellular matrix-free generation of murine testicular organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Organoid generation was considered unsuccessful if testicular cells did not self-assemble within 72 h of culture, however, all methods presented here assemble within 24 h when using juvenile (5 dpp) murine cells. Failure of biological construct generation presented as a continuation of freely suspended cells (0 h column in Figure 1) even after extended culture (72 h). In the absence of tissue self-assembly, any apparent cell clusters easily dispersed into individual cells upon even gentle ma...

Watch this Scientific Journal Video about Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids at JoVE.com

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

These protocols enable the user to produce a large number of structurally mimetic and endocrine functional testicular organoids in both ECM and DCM-free environments in 2D or 3D culture conditions. These techniques are highly reproducible, easily applied to standard biological laboratory settings, and only utilize common tools and materials. Testicular organoids are a novel tool for simulating spermatogenesis and reproductive endocrinology in vitro, and might one day enable the study of in vitro gametogenesis.Begin by placing the euthanized mouse supine on a dissection mat and sterilizing the abdomen with 70%ethanol. Tent the skin of the lower abdomen with forceps and open it with scissors. Locate the testes in the lower left and right inguinal regions of the abdomen and cut their connections to the vas deferens and any anchoring connective tissue.Then lift the entire testes from the animal. Place it in a Petri dish of pre-equilibrated BM.Under a dissection microscope, make a small incision in the tunica albuginea on one end of each testis with either small micro dissection scissors or by gently tearing with two fine forceps. While holding the testis from the opposite end of the incision, gently squeeze it with fine forceps and push in a sweeping motion towards the hole in the tunica, which will release the testicular tissue as one cohesive piece.Cut the tissue into smaller pieces. Place the pieces into one milliliter of prewarm dissociation solution one and incubate them at 37 degrees Celsius for 10 minutes. After the incubation, gently triturate the testes pieces 50 times with a P1000 pipette.Ensure that the tubules separate from one another and from the interstitial tissue. If clumps remain, incubate for an additional five minutes and triturate again. Add 33 microliters of hyaluronidase stock solution per one milliliter of the dissociation mixture.Then triturate 50 times with a P1000 pipette and incubate the sample at 37 degrees Celsius for five minutes. After the incubation, triturate it 50 times with a P200 pipette. After ensuring that no visible tubules or clumps of cells are present, quench the dissociation enzymes by adding FBS to 10%of the total volume of the sample and triturate it several times with a P200 pipette.Wet the center of a 40 micrometer cell strainer with media and aspirate it away, then add the sample to the pre-wetted strainer and filter it to produce a single-cell suspension. Centrifuge the suspension at 100 times g for seven minutes. Discard the supernatant and resuspend the cells in fresh BM.Count the viable cells on a hemocytometer using trypan blue exclusion, then re-centrifuge and resuspend them in fresh BM.For 2D ECM-free culture, plate single-cell suspensions directly onto four-well chamber slides and place the slides into a 35 degree Celsius incubator.For 2D ECM-culture, dispense 100 microliters of cold one-to-one diluted extracellular matrix medium into a four-well chamber slide, ensuring that the gel covers the dish bottom. Place the slide in the incubator for a minimum of 30 minutes to allow the ECM to polymerize into a gel, then add the cell suspension on top. For a 3D ECM-free culture, place agarose 3D Petri dishes into a 24-well culture dish and cover them with one milliliter of BM.Let the dishes equilibrate in BM for at least 30 minutes in a 35 degree Celsius incubator, then discard the BM and repeat the equilibration once more with one milliliter of fresh BM.Remove all BM from the well and dispense 200 microliters of fresh BM around but not inside the center recess of the 3D Petri dish, then collect any remaining BM from inside the center cell seeding recess of the micro well insert.Dispense the single-cell suspension into the center recess and gently triturate up and down. Place the dish into a humidified 35 degree Celsius incubator for culture. On the next day, slowly and carefully remove existing media from around the agarose 3D Petri dish and replace with one milliliter of fresh BM.The organoids should have compacted overnight, allowing them to rest at the bottom.For 3D ECM culture, prepare a single-cell suspension by combining equal parts of the cell suspension in BM with cold pre-thawed ECM. Triturate several times to ensure it is well mixed. Then immediately dispense the mixture into a four-well chamber slide, ensuring that it covers the entire bottom of the plate.Incubate the chamber slides at 35 degrees Celsius and allow its contents to polymerize for at least 30 minutes. After the polymerization, add 500 microliters of BM on top of the culture. Culture all organoid model types at 35 degrees Celsius.For all culture types, exchange half of their media with fresh BM every two days. This protocol was used to achieve organoid generation within 24 hours using juvenile murine testicular cells. Successfully generated tissues were initially observed as 3D cell clusters.In ECM environments, the cell clusters possessed clear margins between the cluster and their surrounding environment. Cell clusters were also observed to migrate across the ECM and fuse together. The 3D ECM culture required significantly more time to assemble de novo structures than all other culture methods.And the 3D ECM-free culture produced the largest clusters with a single large and compact cluster within each well of the 3D agarose Petri dish. To assess whether the organoid models resemble the native tissue, the biological constructs were probed for cell-specific markers and visualized with immunofluorescence. The 2D ECM and 3D ECM-free methods produced organoids with an inner morphology highly mimetic of testicular compartmentalization.A long-term analysis was performed on the 3D ECM-free assembled organoids. The organoids were cultured for 14 days and then probed for cell and structure-specific markers. Tubule-like structures were observed.The 3D ECM-free organoids were also studied for endocrine function in a 12-week culture with gonadotropin stimulation. Testosterone and inhibin B were both quantified from organoid-conditioned medium. After 12 weeks, gonadotropins were removed for 48 hours, causing testosterone and inhibin B concentrations to decrease.When the gonadotropins were returned, both hormone concentrations significantly increased, demonstrating proper endocrine responsiveness. Organoid assembly takes advantage of the autonomous behavior of viable immature testicular cells. Creative experiments can be easily devised through using customized cell sorted or hybrid cell mixtures.Following cell seeding, live video imaging or time-lapse imaging can be used to quantify organoid self-assembly. Across culture, organoids and condition medium can be collected for histological and amino assay analyses.

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6.7K visualizações.  Northwestern University. Esses protocolos permitem ao usuário produzir um grande número de organoides testiculares funcionais estruturalmente miméticos e endócrinos em ambientes livres de ECM e DCM em condições de cultura 2D ou 3D. Essas técnicas são altamente reprodutíveis, facilmente aplicadas a configurações de laboratório biológicos padrão, e utilizam apenas ferramentas e materiais comuns. Organoides testiculares são uma nova ferramenta para simular endocrinologia endocrinologia reprodutiva in vitr...