Method Article

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

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DOI:

10.3791/61403

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October 7th, 2020

 ,  , 
1Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University

In This Article

Summary

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.

Abstract

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.

Introduction

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 “in vitro”, 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’s reliance upon pre-existing native testis tubules, these recent advances are more accurately described as examples of “ex vivo” testicular function and spermatogenesis, tissue function that was maintained or resumed upon removal from an organism’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’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’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.

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Protocol

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

  1. Use two different enzymatic solutions (Solution 1 and Solution 2), both made using a basal culture medium solution (BM).
  2. 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 μm 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 °C for a minimum of 1 h.
    NOTE: BM can be stored at 4 °C for up to 1 week, after which fresh BM should be made.
  3. 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 μL aliquots at -20 °C for later use. Aliquots should be thawed only once.
  4. 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 μL aliquots at -20 °C for later use. Aliquots should be thawed only once.
  5. 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 μL aliquots at -20 °C for later use. Aliquots can be thawed and re-frozen several times without loss of enzymatic activity.
  6. To prepare dissociation Solution 1, add 10 μL collagenase I and 10 μL DNase I into 1 mL of sterile, pre-equilibrated BM (Final concentrations: 1 mg/mL collagenase I and 5 μg/mL DNase I). Triturate gently with a pipette to mix the solution, and pre-warm to 37 °C before use with the tissue.
    NOTE: Solution 2 is prepared by adding 33 μL of hyaluronidase (prewarmed to 37 °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.

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.

  1. 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.
  2. 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.
  3. 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.
    1. 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.
  4. Cut the testes into smaller pieces (≤ 2 mm3) and place them into 1 mL of pre-warmed (37 °C) dissociation Solution 1.
    1. Incubate at 37 °C for 10 min.
    2. 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.).
    3. 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).
  5. Add 33 μL of hyaluronidase stock solution (pre-warmed at 37 °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.
    1. Triturate (50x) using a P1000 and incubate at 37 °C for 5 min.
    2. Triturate (50x) using a P200 pipette.
    3. 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).
  6. 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 μm cell strainer to produce a single-cell suspension.
  7. Centrifuge cells at 100 x g for 7 min, discard the supernatant, and resuspend the cells in fresh BM.
  8. Count the total and viable cell concentrations using trypan blue exclusion on a hemocytometer. Add 10 μL of 1:1 diluted, cell suspension: trypan blue solution, into the hemocytometer cell counting chamber (see Table of Materials).
    1. 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 ≥ 80% viability for generating organoids.
    2. 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 – 0.56 x 106 cells/mL, 2D ECM – 0.56 x 106 cells/mL , 3D ECM-Free – 4.66 x 106 cells/mL, 3D ECM – 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.

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 °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.

  1. For 2D ECM-free culture, no special preparation is necessary, plate single cell suspensions (500 μL of 0.56 x 106 cells/mL in BM) directly onto 4- well chamber slides, and place into a 35 °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.
  2. For 2D ECM culture, dispense 100 μL of cold 1:1 diluted extracellular basement matrix medium into a 4 well chamber slide, ensuring the gel covers the entirety of the dish bottom.
    1. Place the chamber slide in a 35 °C incubator for a minimum of 30 min to allow the ECM to polymerize into a gel.
    2. Add the cell suspension (500 μL 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.
  3. For 3D ECM-free culture, prepare agarose 3D Petri dish inserts before starting the cell culture.
    1. 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.
    2. Within a sterile workspace, dispense molten agarose into the 3D Petri dish mold until the meniscus is level with the sides of the mold.
    3. 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 °C for upwards of one month.
    4. 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 °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).
    5. To prepare for cell seeding, remove all BM from the well and dispense 200 μL 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.
    6. Dispense the single cell suspension (4.66 cells/mL in 60 μL 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.
    7. Place into in a humidified 35 °C incubator for culture. The following day, remove the 200 μL 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.
    8. 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.
  4. 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).
    1. Immediately dispense the cell-ECM mixture into a 4 well chamber slide, ensuring the mixture covers the entire bottom of the plate.
    2. Place the chamber slides at 35 °C in an incubator and allow its contents to polymerize. This should take at least 30 min. After the polymerization, add 500 μL 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.

4. Organoid maintenance

  1. Culture all organoid model types at 35 °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 °C for use with immunoassays or other analyses later (i.e., quantification of secreted reproductive hormones or cytokines).
  2. 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.
  3. 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.

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 °C.

  1. For 2D ECM-free cultures, first rinse the sample with fresh PBS, and then add fixative directly on top of the adhered constructs.
  2. 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.
  3. 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.
  4. Before processing into paraffin, embed many organoids (≥ 20) within a small volume (~ 30 μL) 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.

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Results

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...

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Discussion

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...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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’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’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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.22 um Media Sterile FiltersMillipore Sigmascgpu05reFor sterile filtering media
3βHSD primary antibodyCosmo Bio CoK0607Leydig cell marker, 1:500 dilution
AlexaFluor 568 α-MouseThermo Fisher ScientificA-21202Fluorescence-tagged secondary antibody
AlexaFluor 568 α-RabbitThermo Fisher ScientificA10042Fluorescence-tagged secondary antibody
Alpha Minimum Essential MediumThermo Fisher Scientific11-095-080Base of culture media
Collagenase IWorthington BioLS004197For dissociation solution 1
Corning Matrigel Membrane Matrix, LDEV-freeCorning354234Extracellular matrix used for casting 2D and 3D ECM culture gels
Countess Cell counterThermo Fisher ScientificC10227Autmated cell counter (hemacytometer machine)
Countess Cell Counting Chamber SlidesThermo Fisher ScientificC10228Hemacytometer slide for use with Countess automated counter
DDX4 primary antibodyAbcam138540Spermatogonia marker, 1:500 dilution
Deoxyribonuclease I (2,280 u/mgDW)Worthington BioLS002140For dissociation solution 1
DPBS 1X, + CaCl + MgClThermo Fisher Scientific14040182For reconstituting Hyaluronidase
Dulbecco's Phosphate Buffered Saline +Ca/+MgThermo Fisher Scientific14040117PBS
Embryo Grade H2OMIllipore SigmaW1503For reconstituting Collagenase I and Dnase I
Fetal Bovine SerumThermo Fisher Scientific16000044For quencing enzyme dissocation solutions
Follicle stimulating hormoneAbcamab51888For long-term organoid culture
Human chorionic gonadotropinMillipore SigmaC1063For long-term organoid culture
Hyaluronidase, from bovine testesMillipore SigmaH4272For dissociation solution 2
Inhibin B Enzyme-linked Immunosorbent AssayAnsh LabsAL-107Inhibin B ELISA Kit
KnockOut Serum ReplacementThermo Fisher Scientific10828-028Serum source for Basal media
MicroTissues 3D Petri Dish micro-mold spheroids (24-35, 5x7 array)Millipore SigmaZ764051For 3D ECM-Free organoid fabrication
Nunc, Lab Tek II Chamber Slide System, 4-wellThermo Fisher Scientific12-565-7For 2D ECM-free, and 2D, 3D ECM culture
Penicillin/StreptomycinThermo Fisher Scientific15-140-122Antibiotic for media
Richard-Allan Scientific; Histogel, Specimen processing gelThermo Fisher ScientificHG-4000-012For aiding paraffin embedding
SOX9 primary antibodyMillipore SigmaAB5535Sertoli Marker, 1:500 dilution
Tedklad Global Mouse Chow (Breeder)Teklad Global2920Mouse food without phytoestrogens
Tedklad Global Mouse Chow (Maintenance)Teklad Global2916Mouse food without phytoestrogens
Testosterone Enzyme-linked Immunosorbent AssayCalbiotechTE373STestosterone ELISA Kit
Trypan Blue Solution, 0.4%Thermo Fisher Scientific15250061For cell counting
αSMA primary antibodyMillipore SigmaA2547Peritubular marker, 1:500 dilution
βCatenin primary antibodyBD Biosciences610154Sertoli Cytoplasm marker, 1:100 dilution

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ECM Culture2D ECM free3D ECM freeCellular Self assemblySertoli CellsLeydig CellsGerm CellsPeritubular CellsTissue Architecture

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