Name: extra cellular matrix-based and extra cellular matrix-free generation of murine testicular organoids
Uploaded: 2020-10-07T14:00:00.000+00:00
Duration: 9 min 18 s
Description: Watch this Scientific Journal Video about Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids at JoVE.com

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 10% and 1% respectively (see Table of Materials for specific reagents). Then sterile filter the BM through a 0.22 &#956;m filter. Before use with cells, pre-equilibrate sterile BM to a neutral pH by dispensing into a culture dish and placing within a humidified, 5% CO2 incubator at 37 &#176;C for a minimum of 1 h. 
	NOTE: BM can be stored at 4 &#176;C for up to 1 week, after which fresh BM should be made.</li>
	<li>To prepare collagenase I stock solutions, first dissolve 100 mg of collagenase I into 1 mL of sterile embryo grade H2O (final concentration 10% m/v), invert or swirl to dissolve, and store 20 &#956;L aliquots at -20 &#176;C for later use. Aliquots should be thawed only once.</li>
	<li>To prepare deoxyribonuclease I (DNase I) stock solutions, add 20 mg of DNase I into 1 mL of sterile embryo grade H2O (final concentration 2% m/v), invert or swirl to dissolve (do not vortex), and store 20 &#956;L aliquots at -20 &#176;C for later use. Aliquots should be thawed only once.</li>
	<li>For hyaluronidase stock solutions, add 30 mg into 1 mL of sterile phosphate buffered saline (PBS; final concentration 3% m/v hyaluronidase in PBS containing Ca++/Mg++), invert or swirl to dissolve, and store 100 &#956;L aliquots at -20 &#176;C for later use. Aliquots can be thawed and re-frozen several times without loss of enzymatic activity.</li>
	<li>To prepare dissociation Solution 1, add 10 &#956;L collagenase I and 10 &#956;L DNase I into 1 mL of sterile, pre-equilibrated BM (Final concentrations: 1 mg/mL collagenase I and 5 &#956;g/mL DNase I). Triturate gently with a pipette to mix the solution, and pre-warm to 37 &#176;C before use with the tissue. 
	NOTE: Solution 2 is prepared by adding 33 &#956;L of hyaluronidase (prewarmed to 37 &#176;C) per 1 mL of Solution 1, (to a final concentration of 1 mg/mL). This occurs mid-way through enzymatic dissociation of testis tissue at step 2.5 below.</li>
</ol>
2. Testis tissue dissociation
NOTE: All mice were housed within polypropylene cages and provided with food and water ad libitum. Animals were fed irradiated chow which does not contain phytoestrogens. Juvenile CD-1 mice, 5 days post-partum (dpp), were used for all experiments and anesthetized prior to euthanasia and tissue collection, within an anesthesia chamber attached to an isoflurane vaporizer (2.5 L/min in O2). Mice were confirmed for full anesthesia via the absence of a response to toe-prick, after which mice were euthanized via decapitation.
<ol>
	<li>Anesthetize mice in an isoflurane chamber, ensure anesthesia via a toe-prick, and then decapitate the mouse using a sharp scissor. Place the euthanized mouse supine on a dissection mat and sterilize the abdomen with 70% ethanol. Tent the skin of the lower abdomen with forceps and open the abdomen with scissors.</li>
	<li>Locate the testes in the lower left and right inguinal regions of the abdomen. Cut their connections to the vas deferens and any anchoring connective tissue, then lift the entire testis (with epididymis still attached) from the animal. Place testes in a Petri dish of pre-equilibrated BM.</li>
	<li>Under a dissection microscope and within a sterile field, make a small incision in the tunica albuginea on one end of each testis with either a small microdissection scissor or by tearing gently using two fine forceps.
	<ol>
		<li>Then, while holding the testis from the opposite end of the incision, gently squeeze the testis with fine forceps and push in a gentle sweeping motion towards the hole in the tunica; this will release the testicular tissue as one cohesive piece.</li>
	</ol>
	</li>
	<li>Cut the testes into smaller pieces (&#8804; 2 mm3) and place them into 1 mL of pre-warmed (37 &#176;C) dissociation Solution 1.
	<ol>
		<li>Incubate at 37 &#176;C for 10 min.</li>
		<li>For more than 10 testes, increase the total dissociation solution volume by 1 mL, ensuring a minimum of 1 mL of dissociation solution per 10 testes (e.g., 2 mL for 20 testes, 3 mL for 30 testes, etc.).</li>
		<li>Gently triturate the testis pieces 50 times (50x) in solution 1 using a P1000 pipette. Ensure that the tubules separate from one another and from interstitial tissue at this point. If clumps remain, incubate for an additional 5 min and triturate once more (50x).</li>
	</ol>
	</li>
	<li>Add 33 &#956;L of hyaluronidase stock solution (pre-warmed at 37 &#176;C, from step 1.5) per 1 mL of solution 1 dissociation mixture (containing the partially dissociated testicular tissue and tubules). After adding hyaluronidase, this is called solution 2.
	<ol>
		<li>Triturate (50x) using a P1000 and incubate at 37 &#176;C for 5 min.</li>
		<li>Triturate (50x) using a P200 pipette.</li>
		<li>Ensure that at this point no visible tubules or clumps of cells are present. If clumps persist, incubate for up to 5 more min, with further trituration using a P200 pipette (50x).</li>
	</ol>
	</li>
	<li>Quench the dissociation enzymes by adding fetal bovine serum (FBS) to 10% of the total volume of solution 2. Triturate several times using a P200 pipette to ensure no clumps remain, and filter through a 40 &#956;m cell strainer to produce a single-cell suspension.</li>
	<li>Centrifuge cells at 100 x g for 7 min, discard the supernatant, and resuspend the cells in fresh BM.</li>
	<li>Count the total and viable cell concentrations using trypan blue exclusion on a hemocytometer. Add 10 &#956;L of 1:1 diluted, cell suspension: trypan blue solution, into the hemocytometer cell counting chamber (see Table of Materials).
	<ol>
		<li>Re-centrifuge cells at 100 x g for 7 min and resuspend in fresh BM. 
		NOTE: Only use viable cells for calculating cell concentration and number. Only use cell suspensions of &#8805; 80% viability for generating organoids.</li>
		<li>Prepare the single cell suspension into cell concentrations as described in order to aliquot 280,000 cells given the volumes used in the protocol specific steps below in section 3: 2D ECM-Free &#8211; 0.56 x 106 cells/mL, 2D ECM &#8211; 0.56 x 106 cells/mL , 3D ECM-Free &#8211; 4.66 x 106 cells/mL, 3D ECM &#8211; 2.8 x 106 cells/mL. 
		NOTE: All culture experiments presented here start with 280,000 cells seeded per culture well. These numbers are matched to the representative data in Figure 1, Figure 2, Figure 3 and Figure 4.</li>
	</ol>
	</li>
</ol>
3. Preparation of organoid culture dishes and seeding of cells
NOTE: To ensure a homogenous ECM, pre-thaw frozen aliquots of ECM overnight before experimentation. ECM aliquots should be submerged within a bucket of ice within a 4 &#176;C refrigerator or cold room to guarantee a slow, gradual increase in temperature. All ECM is used at a 1:1 final dilution in BM for culture. Keep thawed ECM and 1:1 diluted ECM on ice until immediately before use, otherwise the ECM might polymerize prematurely.
<ol>
	<li>For 2D ECM-free culture, no special preparation is necessary, plate single cell suspensions (500 &#956;L of 0.56 x 106 cells/mL in BM) directly onto 4- well chamber slides, and place into a 35 &#176;C incubator for culture. 
	NOTE: Cells should adhere to the bottom of the culture dish within the first 24 h of culture and may exhibit some small 3D cell clusters within this same time.</li>
	<li>For 2D ECM culture, dispense 100 &#956;L of cold 1:1 diluted extracellular basement matrix medium&#160;into a 4 well chamber slide, ensuring the gel covers the entirety of the dish bottom.
	<ol>
		<li>Place the chamber slide in a 35 &#176;C incubator for a minimum of 30 min to allow the ECM to polymerize into a gel.</li>
		<li>Add the cell suspension (500 &#956;L of 0.56 x 106 cells/mL in BM) directly on the top of the 2D gel once it has polymerized. 
		NOTE: Cells should cluster together to form small 3D clusters within the first 24 h of culture.</li>
	</ol>
	</li>
	<li>For 3D ECM-free culture, prepare agarose 3D Petri dish inserts before starting the cell culture.
	<ol>
		<li>First, autoclave 1.5 g agarose powder in a 100 mL beaker, then add 75 mL sterile, distilled water and microwave to produce molten 2% agarose for 3D Petri dish casting.</li>
		<li>Within a sterile workspace, dispense molten agarose into the 3D Petri dish mold until the meniscus is level with the sides of the mold.</li>
		<li>Allow the agarose to cool and solidify. When solid, turn the mold upside down and gently flex repeatedly until the agarose 3D Petri dish falls free from the mold. 
		NOTE: At this point, one can prepare many agarose 3D Petri dishes and store them in sterile H2O or DPBS at 4 &#176;C for upwards of one month.</li>
		<li>Prior to culturing, place agarose 3D Petri dishes into a 24 well culture dish, and cover them with 1 mL of BM. Let the 3D Petri dishes equilibrate in BM for at least 30 min within a 37 &#176;C culture incubator. Discard the BM, and repeat the equilibration once more with 1 mL fresh BM. After equilibration of 3D Petri dishes in BM, they will appear the same color as the BM (i.e., pink).</li>
		<li>To prepare for cell seeding, remove all BM from the well and dispense 200 &#956;L of fresh BM around, but not inside the center recess of the 3D Petri dish. Also, collect any remaining BM from inside the center cell-seeding recess of the microwell insert.</li>
		<li>Dispense the single cell suspension (4.66 cells/mL in 60 &#956;L of BM) into the center recess of the agarose 3D Petri dish. Gently triturate up and down to mix cells and guarantee a single cell suspension at the start of culture.</li>
		<li>Place into in a humidified 35 &#176;C incubator for culture. The following day, remove the 200 &#956;L of BM from around the microwell insert, and replace with 1 mL of fresh BM. This will bring the liquid level above the plane of the insert, submerging the entire culture.</li>
		<li>Slowly and carefully remove/add media from outside of the agarose 3D Petri dish. The organoids should have compacted overnight, allowing them to rest at the bottom and enabling media changes to leave organoids undisturbed.</li>
	</ol>
	</li>
	<li>For 3D ECM culture, prepare a single cell suspension by combining, in equal parts, the cell suspension in BM with cold, pre-thawed ECM (final concentration = 2.8 x 106 cells/mL).
	<ol>
		<li>Immediately dispense the cell-ECM mixture into a 4 well chamber slide, ensuring the mixture covers the entire bottom of the plate.</li>
		<li>Place the chamber slides at 35 &#176;C in an incubator and allow its contents to polymerize. This should take at least 30 min. After the polymerization, add 500 &#956;L of BM on the top of the culture. 
		NOTE: Cells should have clustered together to form small 3D aggregates within the first 24 h of culture.</li>
	</ol>
	</li>
</ol>
4. Organoid maintenance
<ol>
	<li>Culture all organoid model types at 35 &#176;C. For All culture types exchange half of their media with fresh BM every 2 days. To ensure that organoids are not accidentally collected while exchanging medium, always collect media slowly from a corner of the chamber slide dish, and from an external point outside of agarose 3D Petri dishes. All media can be stored at -20 &#176;C for use with immunoassays or other analyses later (i.e., quantification of secreted reproductive hormones or cytokines).</li>
	<li>After 7 days in culture, use BM containing follicle stimulating hormone (final concentration 20 mIU/mL) and human chorionic gonadotropin (final concentration 4.5 IU/mL). This applies to all organoid culture types.</li>
	<li>Routinely image all organoid cultures (i.e., time-lapse imaging) for characterizing organoid formation and quantifying metrics of self-assembly, development, and growth over time.</li>
</ol>
5. Organoid Collection
NOTE: All organoids can be fixed with 4% paraformaldehyde in PBS for downstream immunolabeling and histological analyses. Fix for 2 h at room temperature with rotation, or overnight at 4 &#176;C.
<ol>
	<li>For 2D ECM-free cultures, first rinse the sample with fresh PBS, and then add fixative directly on top of the adhered constructs.</li>
	<li>For ECM (2D and 3D) culture methods, rinse once with PBS, and then either add fixative directly on the top of the ECM-organoid sample (to fix the ECM gel and organoids together), or alternatively, gently pipette the organoids up and down to free them from the surrounding ECM, and transfer to a separate tube for fixation.</li>
	<li>For 3D ECM-free culture, gently pipette the organoids up and down within the center recess of the agarose 3D Petri dish; this will flush the organoids out facilitating their easy collection with a pipette. Then transfer organoids to a separate tube for fixation.</li>
	<li>Before processing into paraffin, embed many organoids (&#8805; 20) within a small volume (~ 30 &#956;L) of tissue processing gel; this helps orient and concentrate organoids into a small area within paraffin blocks, facilitating easier observation when sectioning and easier visual identification within paraffin sections. 
	NOTE: Organoids can be challenging to identify after paraffin embedding and sectioning upon a microtome.</li>
</ol>

The authors have nothing to disclose.

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&amp;beta;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 &amp;alpha;-Mouse</td><td>Thermo Fisher Scientific</td><td>A-21202</td><td>Fluorescence-tagged secondary antibody</td></tr><tr><td>AlexaFluor 568 &amp;alpha;-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&#039;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>&amp;alpha;SMA primary antibody</td><td>Millipore Sigma</td><td>A2547</td><td>Peritubular marker, 1:500 dilution</td></tr><tr><td>&amp;beta;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 ...

extra-cellular-matrix-based-extra-cellular-matrix-free-generation

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

6.8K Views.  Northwestern University. 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.

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

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

Genetics

Prostate Organoid Cultures as Tools to Translate Genotypes and Mutational Profiles to Pharmacological Responses

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate organoids are androgen responsive, three-dimensional (3D) cultures grown in a defined medium that resembles the prostatic epithelium. Prostate organoids can be established from wild-type and genetically engineered mouse models, benign human tissue, and advanced prostate cancer. Importantly, patient derived organoids closely resemble tumors in genetics and in vivo tumor biology. Moreover, organoids can be genetically manipulated using CRISPR/Cas9 and shRNA systems. These controlled genetics make the organoid culture attractive as a platform for rapidly testing the effects of genotypes and mutational profiles on pharmacological responses. However, experimental protocols must be specifically adapted to the 3D nature of organoid cultures to obtain reproducible results. Described here are detailed protocols for performing seeding assays to determine organoid formation capacity. Subsequently, this report shows how to perform drug treatments and analyze pharmacological response via viability measurements, protein isolation, and RNA isolation. Finally, the protocol describes how to prepare organoids for xenografting and subsequent in vivo growth assays using subcutaneous grafting. These protocols yield highly reproducible data and are widely applicable to 3D culture systems.

Presented here is a protocol to study pharmacological responses in prostate epithelial organoids. Organoids closely resemble in vivo biology and recapitulate patient genetics, making them attractive model systems. Prostate organoids can be established from wildtype prostates, genetically engineered mouse models, benign human tissue, and advanced prostate cancer.

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate ...

Cancer Research

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Generation of Multicellular Human Primary Endometrial Organoids

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

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

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

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Basement Membrane Matrix Encapsulated Cell Aggregation for Investigating Murine Spleen Tissue Formation

The spleen is an immune organ that plays a key role in blood-borne immune responses. The anatomical or functional loss of this tissue increases susceptibility to severe blood infections and sepsis. Auto-transplantation of spleen slices has been used clinically to replace lost tissue and restore immune function. However, the mechanism driving robust and immunologically functional spleen tissue regeneration has not been fully elucidated. Here, we aim to develop a method for aggregating and encapsulating spleen cells within a semi-solid matrix in order to investigate the cellular requirements for spleen tissue formation. Basement membrane matrix encapsulated cell constructs are amenable to both in vitro tissue culture of three-dimensional organoids as well as transplantation under the kidney capsule to directly assess in vivo tissue formation. By manipulating the input cells for aggregation and encapsulation, we demonstrate that graft-derived PDGFR&#946;+MAdCAM-1- neonatal stromal cells are required for spleen tissue regeneration under animal transplantation models.

This article describes a protocol for aggregating and encapsulating spleen cells within a semi-solid basement membrane matrix. Basement membrane matrix constructs can be used in three-dimensional culture for studying organoid development, or for in vivo transplantation and tissue regeneration studies.

The spleen is an immune organ that plays a key role in blood-borne immune responses. The anatomical or functional loss of this tissue increases ...

Matrix Comparison for Efficient Human Intestinal Organoid Generation from iPSCs

Comparative Study of Basement-Membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.

Author Spotlight: Advancing Organoid Generation for Drug Development Using iPSCs

Organoids have become valuable tools for disease modeling. The extracellular matrix (ECM)&#160;guides cell fate during organoid generation, and using a system that resembles the native tissue can improve model accuracy. This study compares the generation of induced pluripotent stem cells-derived human intestinal organoids in animal-derived ECM and xeno-free hydrogels.

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) ...

Bioengineering

Three-Step Organoid Model for Tendon Research and Engineering

Demonstration of Self-Assembled Cell Sheet Culture and Manual Generation of a 3D Tendon/Ligament-Like Organoid by using Human Dermal Fibroblasts

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting&#160;the&#160;musculoskeletal system. These tissues have a strictly arranged collagen type&#160;I-rich extracellular matrix (ECM) and T/L-lineage cells mainly positioned in parallel rows. After injury, T/L require a long time for rehabilitation with high failure risk and often unsatisfactory repair outcomes. Despite recent advancements in T/L biology research, one of the remaining challenges is that the T/L field still lacks a standardized differentiation protocol that is able to recapitulate T/L formation process in vitro. For example, bone and fat differentiation of mesenchymal precursor cells require just standard two-dimensional (2D) cell culture and the addition of specific stimulation media. For differentiation to cartilage, three-dimensional (3D) pellet culture and supplementation of TGF&#223; is necessary. However, cell differentiation to tendon needs a very orderly 3D culture model, which ideally should also be subjectable to dynamic mechanical stimulation. We have established a 3-step (expansion, stimulation, and maturation) organoid model to form a 3D rod-like structure out of a self-assembled cell sheet, which delivers a natural microenvironment with its own ECM, autocrine, and paracrine factors. These rod-like organoids have a multi-layered cellular architecture within rich ECM and can be handled quite easily for exposure to static mechanical strain. Here, we demonstrated the 3-step protocol by using commercially available dermal fibroblasts. We could show that this cell type forms robust and ECM-abundant organoids. The described procedure can be further optimized in terms of culture media and optimized toward dynamic axial mechanical stimulation. In the same way, alternative cell sources can be tested for their potential to form T/L organoids and thus undergo T/L differentiation. In sum, the established 3D T/L organoid approach can be used as a model for tendon basic research and even for scaffold-free T/L engineering.

Author Spotlight: Advancing Tendon Tissue Engineering with 3D Organoid Models

Herein, we demonstrate a three-step organoid model (two-dimensional [2D] expansion, 2D stimulation, three-dimensional [3D] maturation) offering a promising tool for tendon fundamental research and a potential scaffold-free method for tendon tissue engineering.

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting&#160;the&#160;musculoskeletal system. These tissues have a strictly ...

Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs in vivo. Formation of organoids has opened up different avenues of basic and translational research. In recent years, testicular organoids have garnered interest in the field of male reproductive biology. Testicular organoids allow for the study of cell-cell interactions, tissue development, and the germ cell niche microenvironment and facilitate high throughput drug and toxicity screening. A method is needed to reliably and reproducibly generate testicular organoids with testis specific tissue architecture. The microwell culture system contains a dense array of pyramid-shaped microwells. Testicular cells derived from pre-pubertal testes are centrifuged into these microwells and cultured to generate testicular organoids with testis-specific tissue architecture and cell associations. Thousands of homogeneous organoids can be generated via this process. The protocol reported here will be of broad interest to researchers studying male reproduction.

Here, we present a protocol for the reproducible generation of porcine testicular organoids with testis specific tissue architecture using the commercially available microwell culture system.

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs ...