Name: Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture (Video) | JoVE Journal
Uploaded: 2019-10-03T15:00:00.000+00:00
Duration: 11 min 53 s
Description: Learn to generate porcine testicular organoids with testis-specific architecture using microwell culture. This reproducible method enables studying testis morphogenesis and high-throughput drug screening applications.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by NIH/NICHD HD091068-01 to Dr. Ina Dobrinski.

NOTE: Testes from 1-week-old piglets were obtained from a commercial pig farm as by-product from castration of commercial pigs. Sourcing of testes was approved by the Animal Care Committee at the University of Calgary.
1. Preparation of enzyme solutions for tissue digestion
NOTE: Three different enzymatic solutions are needed, which include two different collagenase IV solutions (solution A, B) and a deoxyribonuclease I (DNase I) solution.
<ol>
	<li>To prepare solution A, dissolve 20 mg of collagenase IV S (Table of Materials) and 40 mg of collagenase IV W (Table of Materials) in 25 mL of high glucose Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM). Then filter sterilize with a 0.22 &#181;m filter. Add 0.4 mL of fetal bovine serum (FBS) to solution A to inhibit trypsin activity.</li>
	<li>To prepare solution B, dissolve 80 mg of collagenase IV W (Table of Materials) in 40 mL of DMEM. Then filter sterilize with a 0.22 &#181;m filter.</li>
	<li>To prepare DNase I solution (7 mg/mL), dissolve 70 mg of DNase I in 10 mL of DMEM. Then filter sterilize with a 0.22 &#181;m filter. 
	NOTE: The enzyme concentration for collagenase IV solutions outlined here, varies from the concentrations (2 mg/mL) stated in the original article14. The current protocol yields cells with slightly higher viability. However, cells isolated by both protocols produce organoids with identical tissue architecture.</li>
</ol>
2. Testis tissue enzymatic digestion
<ol>
	<li>Collect the testes into a sterile beaker and wash with phosphate buffered saline (PBS) containing 1% penicillin/streptomycin (P/S). After washing, transfer the testes to a 100 mm tissue culture dish with PBS containing 1% P/S and remove the tunica vaginalis and epididymis using autoclaved scissors and forceps. Transfer the isolated testes to a new 100 mm dish and wash thoroughly with PBS containing 1% P/S.</li>
	<li>To maintain sterility, use another set of sterile scissors and forceps to peel the testicular parenchyma out of the tunica albuginea. Cut the testes along the longitudinal axis directly under the tunica. Then peel the testes out of the tunica using two forceps and place into a new 100 mm dish containing of 1 mL DMEM with 1% P/S.</li>
	<li>Mince the peeled testes with sterile scissors into 1-2 mm tissue pieces. After chopping, use sterile forceps to remove white fragments of connective tissue.</li>
	<li>Transfer the minced tissue pieces into solution A and top it up to 50 mL with DMEM to obtain a concentration of 0.4 mg/mL for collagenase IV S (Table of Materials) and a concentration of 0.8 mg/mL for collagenase IV W (Table of Materials). Place solution A containing the tissue pieces into a 37 &#176;C water bath for 30 min, gently invert the tubes every 5 min and check visually for the release of DNA.
	<ol>
		<li>Add 500 &#181;L of DNase I if free floating DNA is observed. After 30 min, centrifuge the tube at 90 x g with brakes at 25 &#176;C for 1.5 min and discard the supernatant. 
		NOTE: DNA would appear as a cloudy substance. When the tubes are shaken, the tissue pieces settle down whereas the DNA would stay afloat.</li>
	</ol>
	</li>
	<li>Add solution B to the tube and top up to 50 mL with DMEM to obtain a concentration of 1.2 mg/mL of collagenase IV W (Table of Materials). Place the tube into 37 &#176;C water bath for 30 min and gently invert the tube every 5 min. Add 500 &#181;L of DNase I if free floating DNA is observed.</li>
	<li>After 30 min, centrifuge the tube at 90 x g with brakes at 25 &#176;C for 1.5 min. After that discard the supernatant and wash once with PBS with 1% P/S. 
	NOTE: The discarded supernatants from both solution A and B will primarily contain interstitial cells.</li>
	<li>Top up the tube with PBS up to 50 mL. Carefully collect the tubules from the top and place into a new 50 mL tube. 
	NOTE: The big undigested tissue fragments quickly settle at the bottom, while digested seminiferous tubules will remain in suspension. No big tissue fragments should be collected. This procedure can be repeated several times until the solution in the original tube is almost clear and just undigested tissue fragments are remaining, which can be disposed.</li>
	<li>Centrifuge the tubules at 90 x g with brakes at 25 &#176;C for 1.5 min and discard the supernatant. Add fresh PBS and centrifuge again. Repeat this wash step twice.</li>
	<li>After the last PBS wash, remove the supernatant and resuspend the seminiferous tubules in 5 mL of PBS. Then add 15 mL of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) to the tubules. Place the tube in 37 &#176;C water bath and gently invert every 2 min. If a lot of free-floating DNA is observed, add 500 &#181;L of DNase I with 5 mL of DMEM.</li>
	<li>Evaluate the enzymatic digestion of tubules to single cells under the microscope (first after 5 min, then every 2 min). If mostly single cells can be detected, stop the reaction with 5 mL of FBS and filter through a 70 &#181;m mesh and then through a 40 &#181;m mesh. 
	NOTE: Care should be taken that the caps on the 50 mL tubes used for digestions (Solution A, B and 0.25% trypsin-EDTA) are tightened properly. If contamination in the water bath is a concern, paraffin film may be wrapped around the cap to ensure sterility.</li>
	<li>Centrifuge the single cells at 500 x g with brakes at 25 &#176;C for 5 min, resuspend in the enrichment medium (Dulbecco Modified Eagle Medium F/12 (DMEM/F12) containing 5% FBS and 1% P/S) and count the number of viable cells. This is the starting cell population. Fix 100 x 103 cells and perform immunocytochemistry for germ cell marker UCHL1 (Ubiquitin C-Terminal Hydrolase L1) as described12 and determine the percentage of germ cells in this starting cell population. 
	NOTE: Germ cell marker Promyelocytic leukemia zinc finger protein (PLZF)15 could also be used as a suitable alternative for UCHL1. In the starting cell population, the expected cell yield is around 700-800 x 109/g of tissue. The percentage of germ cells in this cell population should be 4-5%.</li>
	<li>Place around 20 x 106 of this starting cell population in two ultra low attachment 100 mm Petri dishes (10 x 106 cells suspended in 10 mL of DMEM/F12 containing 1% P/S in each dish) for 2 days in a tissue culture incubator (37 &#176;C, 5% CO2, 21% oxygen). Perform the germ cell enrichment with the remaining cells. 
	NOTE: To ensure optimum viability and cell quality, while the germ cell enrichment and subsequent quantification takes place, the starting cell population is placed in culture in ultra low attachment tissue culture dishes for 2 days.</li>
</ol>
3. Germ cell enrichment
NOTE: The procedure described above yields primarily Sertoli cells and germ cells. Different adhesion properties allow for the separation of Sertoli cells and germ cells via differential plating.
<ol>
	<li>Seed 20-25 x 106 cells of the starting cell population per 100 mm tissue culture dish in a total volume of 8 mL of enrichment medium (DMEM/F12 with 5% FBS and 1% P/S) for differential plating. Place the cells into an incubator (37 &#176;C, 5% CO2, 21% O2) and after 1.5 h, ensure that the majority of Sertoli cells attach to the plate.</li>
	<li>Collect and combine the supernatant (mainly containing germ cells) of 2 plates to one new 100 mm plate and place it back into the incubator. After 1 h, again combine the supernatant of 2 plates to a new 100 mm plate. Place the plates back into the incubator for overnight. 
	NOTE: The supernatants combined will primarily contain germ cells. The adhered cells that are discarded along with the plates are primarily testicular somatic cells such as Sertoli and peritubular myoid cells.</li>
	<li>Collect the enriched germ cells in two fractions as follows. 
	NOTE: Enriched germ cells adhere differently, non adherent fraction and slightly adhered fraction. Both these fractions together form the enriched germ cell population
	<ol>
		<li>Non adherent fraction: collect the supernatant.</li>
		<li>Slightly adherent fraction: wash the plates gently with PBS, treat with 2 mL of 1:20 dilution of 0.25% trypsin-EDTA for 5 min at room temperature, stop the reaction with 2 mL of enrichment medium and collect in the same tube as non adherent fraction. 
		NOTE: 0.25% Trypsin-EDTA needs to be diluted with PBS to produce a 1:20 trypsin solution.</li>
	</ol>
	</li>
	<li>Count the total number of cells using a cell counter, fix 100 x 103 cells and perform immunocytochemistry for germ cell marker UCHL1 as described12 and determine the percentage of germ cells in this enriched cell population. 
	NOTE: The percentage of germ cells in this cell population should be at least 60-70%. Since the immunocytochemistry for quantification would require 1 day, the enriched cells should be placed in an ultra low attachment 100 mm Petri dish with DMEM/F12 supplemented with 1% P/S for the duration to ensure optimum cell quality and viability.</li>
</ol>
4. Preparation of cells for seeding
<ol>
	<li>To harvest the starting cell preparation, collect the supernatant in a 50 mL tube from the ultra low attachment 100 mm dishes. Wash the plates vigorously with PBS to collect the adhered cells. No enzymatic digestion is needed. 
	NOTE: Some of the testicular cells, primarily Sertoli cells, can slightly adhere to the ultra low attachment plates.</li>
	<li>Combine the starting cell preparation and enriched germ cells to obtain a working cell preparation containing 25% germ cells. Centrifuge at 500 x g with brakes at 25 &#176;C for 5 min, discard the supernatant and resuspend the cells in organoid formation medium (DMEM/F12 supplemented with insulin 10 &#181;g/mL, transferrin 5.5 &#181;g/mL, selenium 6.7 ng/mL, 20 ng/mL epidermal growth factor, 1% P/S). Adjust the cell density to 2.4 x 106 per mL. 
	NOTE: Each well in the microwell plates used13 in this protocol contained 1,200 microwells.</li>
	<li>Use the formula below to calculate the number of cells per well as described below. 
	Number of cells per well = number of microwells x number of cells to be clustered for each organoid.</li>
	<li>To generate organoids from 1, 000 cells each, seed each with (1,200 x 1,000 cells each=) 1.2 x 106 cells. Adjust the cell density in the cell suspension in a way that the cells are seeded in a volume of 0.5 mL of medium. For organoids formed from 1,000 cells each, use a density of 2.4 x 106 cells per mL (1.2 x 106 cells in 0.5 mL).</li>
</ol>
5. Preparation of microwells to receive cells
NOTE: To ensure that the cells do not adhere to the microwell surface, treat with a surfactant rinsing solution that is available for purchase by the manufacturer.
<ol>
	<li>Add 0.5 mL of rinsing solution to each well. Ensure that no air bubbles are trapped in the well. To remove air bubbles, if any, centrifuge the plate at 2,000 x g with brakes at 25 &#176;C for 2 min.</li>
	<li>Observe the plate under a low-magnification inverted microscope, to verify that the bubbles have been removed from the microwells. If trapped bubbles are observed, centrifuge again at 2,000 x g with brakes at 25 &#176;C for 2 min to remove any remaining bubbles.</li>
	<li>Cover the plate with the lid and incubate for 30 min at room temperature. After the treatment is complete, remove the rinsing solution and immediately wash the plate with sterile water or PBS. Ensure that the plate do not dry out after the treatment with the rinsing solution.</li>
</ol>
6. Generation of testicular organoids
<ol>
	<li>Add 0.5 mL of organoid formation medium (without any cells) to each well and centrifuge at 2,000 x g with brakes at 25 &#176;C for 2 min to remove any trapped air bubbles. Observe the well under a low-magnification inverted microscope to verify that the air bubbles have been removed. If trapped bubbles are observed, centrifuge again at 2,000 x g with brakes at 25 &#176;C for 2 min to remove any remaining bubbles.</li>
	<li>Add 0.5 mL of the working cell suspension and gently mix by pipetting up and down. Centrifuge at 500 x g with brakes at 25 &#176;C for 5 min. Use an inverted microscope to verify that the cells have clustered within the microwells.</li>
	<li>Transfer the plate into a cell culture incubator and culture for 5 days. Change half of the medium every other day.
	<ol>
		<li>To change medium without losing any organoids, touch the pipette tip to the wall of the well and lower gently to touch the meniscus and slowly draw the medium. Make sure to follow the meniscus as it drops down. To add fresh medium, touch the pipette tip to the wall of the well on the same side as the medium withdrawal and add fresh medium gently against the well wall, allowing it to slowly flow down the wall.</li>
	</ol>
	</li>
	<li>To recover the organoids, gently pipette the medium up and down using a wide mouth pipette. This would allow the organoids to come out of the microwells.</li>
	<li>Collect the organoids, wash with PBS, fix and perform immunocytochemistry for germ cell marker (UCH-L1), Sertoli cell marker (GATA Binding Protein 4-GATA4), peritubular myoid cell marker (&#945;-Smooth Muscle Actin-&#945;SMA) and Leydig cell marker (Cytochrome P450-CYP450) as described12 and visualize under a confocal microscope.</li>
</ol>

<ol><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li>
<li>Barker, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lgr5%28%2Bve%29+stem+cells+drive+self-renewal+in+the+stomach+and+build+long-lived+gastric+units+in+vitro%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro.</a> Cell Stem Cell. 6 (1), 25-36 (2010).</li>
<li>Huch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unlimited+in+vitro+expansion+of+adult+bi-potent+pancreas+progenitors+through+the+Lgr5%2FR-spondin+axis%5BTitle%5D)+AND+%22Embo+Journal%22%5BJournal%5D)">Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.</a> Embo Journal. 32 (20), 2708-2721 (2013).</li>
<li>Boj, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li>
<li>Takebe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vascularized+and+functional+human+liver+from+an+iPSC-derived+organ+bud+transplant%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Vascularized and functional human liver from an iPSC-derived organ bud transplant.</a> Nature. 499 (7459), 481-484 (2013).</li>
<li>Quadrato, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+diversity+and+network+dynamics+in+photosensitive+human+brain+organoids%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Cell diversity and network dynamics in photosensitive human brain organoids.</a> Nature. 545 (7652), 48-53 (2017).</li>
<li>Abbott, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+culture%3A+biology%27s+new+dimension%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Cell culture: biology's new dimension.</a> Nature. 424 (6951), 870-872 (2003).</li>
<li>Pendergraft, S. S., Sadri-Ardekani, H., Atala, A., Bishop, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+testicular+organoid%3A+a+novel+tool+for+the+study+of+human+spermatogenesis+and+gonadotoxicity+in+vitrodagger%5BTitle%5D)+AND+%22Biology+of+Reproduction%22%5BJournal%5D)">Three-dimensional testicular organoid: a novel tool for the study of human spermatogenesis and gonadotoxicity in vitrodagger.</a> Biology of Reproduction. 96 (3), 720-732 (2017).</li>
<li>Strange, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+testicular+organoid+system+as+a+novel+tool+to+study+Zika+virus+pathogenesis%5BTitle%5D)+AND+%22Emerging+Microbes+%26+Infections%22%5BJournal%5D)">Human testicular organoid system as a novel tool to study Zika virus pathogenesis.</a> Emerging Microbes & Infections. 7 (1), 82-82 (2018).</li>
<li>Alves-Lopes, J. P., Soder, O., Stukenborg, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Testicular+organoid+generation+by+a+novel+in+vitro+three-layer+gradient+system%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Testicular organoid generation by a novel in vitro three-layer gradient system.</a> Biomaterials. 130, 76-89 (2017).</li>
<li>Baert, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primary+Human+Testicular+Cells+Self-Organize+into+Organoids+with+Testicular+Properties%5BTitle%5D)+AND+%22Stem+Cell+Reports%22%5BJournal%5D)">Primary Human Testicular Cells Self-Organize into Organoids with Testicular Properties.</a> Stem Cell Reports. 8 (1), 30-38 (2017).</li>
<li>Sakib, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Formation+of+organotypic+testicular+organoids+in+microwell+culture%5BTitle%5D)+AND+%22Biology+of+Reproduction%22%5BJournal%5D)">Formation of organotypic testicular organoids in microwell culture.</a> Biology of Reproduction. , (2019).</li>
<li>Razian, G., Yu, Y., Ungrin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+Large+Numbers+of+Size-controlled+Tumor+Spheroids+Using+Microwell+Plates%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates.</a> Journal of Visualized Experiments. (81), 50665(2013).</li>
<li>Sakib, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Formation+of+organotypic+testicular+organoids+in+microwell+culture%5BTitle%5D)+AND+%22Biology+of+Reproduction%22%5BJournal%5D)">Formation of organotypic testicular organoids in microwell culture.</a> Biology of Reproduction. 100 (6), 1648-1660 (2019).</li>
<li>González, R., Dobrinski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Beyond+the+Mouse+Monopoly%3A+Studying+the+Male+Germ+Line+in+Domestic+Animal+Models%5BTitle%5D)+AND+%22ILAR+Journal%22%5BJournal%5D)">Beyond the Mouse Monopoly: Studying the Male Germ Line in Domestic Animal Models.</a> ILAR Journal. 56 (1), 83-98 (2015).</li>
<li>Oatley, J. M., Brinster, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+germline+stem+cell+niche+unit+in+mammalian+testes%5BTitle%5D)+AND+%22Physiological+Reviews%22%5BJournal%5D)">The germline stem cell niche unit in mammalian testes.</a> Physiological Reviews. 92 (2), 577-595 (2012).</li>
<li>Chen, L. Y., Willis, W. D., Eddy, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+the+Gdnf+Gene+in+peritubular+myoid+cells+disrupts+undifferentiated+spermatogonial+cell+development%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Science+USA%22%5BJournal%5D)">Targeting the Gdnf Gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development.</a> Proceedings of the National Academy of Science USA. 113 (7), 1829-1834 (2016).</li>
</ol>

Dr. Ungrin has a financial interest in the AggreWell technology as an inventor.

We have established a simple method that allows the consistent, repeatable generation of large numbers of testicular organoids with tissue architecture that is similar to testis in vivo12. While the approach was developed using porcine testis cells, it is more broadly applicable also to mouse, non-human primate and human testis12. A number of different methods have been reported for producing testicular organoids8,9...

In recent years, there has been a resurgence of interest in three-dimensional (3D) organoids. Different organs such as intestine1, stomach2, pancreas3,4, liver5, and brain6 have been successfully derived into 3D organoid systems. These organoids have architectural and functional similarities to the organs in vivo and are more biologically relevant for study of tissue microenvironment than monolayer culture systems7. As a result, testicular organoids have started to garner interest as well8,9,10,11,12. The majority of methods reported so far are complex, non-high throughput10 and require the addition of ECM proteins8,10. This complexity also leads to issues with reproducibility. A simple and reproducible method is needed that allows for the generation of testicular organoids with cell-associations that are like testis in vivo.
We have recently reported a system to address these requirements12. Using the pig as a model, we employed a centrifugal forced aggregation approach in the microwell system. In the microwell system, each well contains a large number of identical smaller microwells13. This allows for the generation of numerous spheroids of uniform size. The microwell system enabled generation of large numbers of uniform organoids with a testis-specific architecture. The system is simple and does not require addition of ECM proteins.

<table><tbody><tr><td>100 mm ultra low attachment tissue culture dish</td><td>Corning</td><td>#CLS3262</td><td></td></tr><tr><td>100 mm tissue culture dish</td><td>Corning</td><td>#353803</td><td></td></tr><tr><td>Aggrwell 400</td><td>Stemcell Technologies</td><td>#34411</td><td></td></tr><tr><td>Anti-Adherence Rinsing Solution</td><td>Stemcell Technologies</td><td>#07010</td><td></td></tr><tr><td>Collagenase type IV from Clostridium histolyticum</td><td>Sigma-Aldrich</td><td>#C5138</td><td>referred as Collagenase IV S</td></tr><tr><td>Collagenase type IV Worthington</td><td>Worthington-Biochem</td><td>#LS004189</td><td>referred as Collagenase IV W</td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma-Aldrich</td><td>#DN25</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium/F12</td><td>Gibco</td><td>#11330-032</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium - high glucose</td><td>Sigma-Aldrich</td><td>#D6429</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>#D8537</td><td></td></tr><tr><td>Epidermal Growth Factor</td><td>R&amp;amp;D Systems</td><td>#236-EG</td><td></td></tr><tr><td>Falcon Cell Strainers 70 &amp;micro;m</td><td>FisherScientific</td><td>#352350</td><td></td></tr><tr><td>Falcon Cell Strainers 40 &amp;micro;m</td><td>FisherScientific</td><td>#352340</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>ThermoFisher Scientific</td><td>#12483-020</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium</td><td>Gibco</td><td>#41400-045</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>#P4333</td><td></td></tr><tr><td>Porcine testicular tissue</td><td>Sunterra Farms Ltd (Alberta, Canada)</td><td></td><td></td></tr><tr><td>Steriflip-GP Sterile Centrifuge Tube Top Filter Unit</td><td>Millipore</td><td>#SCGP00525</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma</td><td>#T4049</td><td></td></tr></tbody></table>

generation of porcine testicular organoids with testis specific architecture using microwell culture

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.

Isolated cells from 1-week old porcine testes that were cultured in the microwells self-organized into spheroids (Figure 1A, Figure 2), with delineated and distinct exterior (seminiferous epithelium) and interior compartments (interstitium) (Figure 1B, Figure 2). The two compartments were separated by a collagen IV+ve basement...

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 ...

generation-porcine-testicular-organoids-with-testis-specific

Research

JoVE Journal

Medicine

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.

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

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.

Generation of Mosaic Mammary Organoids by Differential Trypsinization

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.

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

This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm2, in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed.

A High-Throughput Platform for Culture and 3D Imaging of Organoids

Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells.

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

This protocol presents a method for generating vascular organoids using human pluripotent stem cells.

Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells

This study outlines a protocol for generating 2D monolayers of porcine organoids derived from the small and large intestines. The growth of these monolayers is marked by increasing TEER values, indicating robust epithelial integrity. Additionally, these monolayers exhibit physiological secretory responses in Ussing chamber experiments following the application of forskolin.

Two-dimensional Porcine Intestinal Organoids Reflecting the Physiological Properties of Native Gut

This study presents a cost-effective and efficient methodology for generating 3D cell structures using a stamp-based system to create microwells in agarose molds. The system promotes the formation of uniform spheroids/organoids, thus improving cell interactions. This approach reduces experimental variability and supports applications in drug testing and tissue engineering.

Generation of Three-Dimensional Spheroids/Organoids from Two-Dimensional Cell Cultures Using a Novel Stamp Device

We describe methods for isolating mouse ovarian somatic cells from a stroma-enriched fraction of the ovary and for generating mouse ovarian somatic organoids using a scaffold-free approach.

Probing the Ovarian Microenvironment: Methods for Isolation, 2D Culture, and Organoid Culture of Mouse Ovarian Somatic Cells

Human pluripotent stem cells (hPSCs) have the potential to treat a myriad of different diseases.  The utility of these cells lies in the fact that they can differentiate into any cell type in the body.  Here we describe the teratoma assay, which is used to demonstrate the pluripotence of hPSCs.

Teratoma Generation in the Testis Capsule

Learn to generate porcine testicular organoids with testis-specific architecture using microwell culture. This reproducible method enables studying testis morphogenesis and high-throughput drug screening applications.

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

Explore More Videos