Microfluidic systems in testicular in vitro culture: a powerful model tool for spermatogenesis and reprotoxicity studies

Abstract

As prepubertal boys do not yet produce spermatozoa, they cannot rely on sperm cryopreservation for fertility preservation before gonadotoxic therapy, such as high-dose alkylating agents or radiotherapy in the case of childhood cancers. According to the current guidelines, cryopreservation of testicular biopsies containing spermatogonial stem cells (SSCs) may be proposed to high-risk patients for potential later therapeutic use to fulfill the patients’ wish for a biological child. One promising technique for human in vitro spermatogenesis and in vitro propagation of human SSCs is microfluidic (MF) culture, in which cells or tissues are subjected to a continuous flow of medium. This provides exact control over such parameters as nutrient content and gradients, as well as the removal of waste metabolites. While MF has been shown to maintain tissues and cell populations of organs for longer than conventional in vitro culture techniques, it has not been widely used for testicular in vitro culture. MF could advance human testicular in vitro culture and is also applicable to reprotoxicity studies. This review summarizes the findings and achievements of testis-on-chip (ToC) setups to date and discusses the benefits and limitations of these for spermatogenesis in vitro and toxicity assessment.

INTRODUCTION

Conventional two-dimensional (2D) and three-dimensional (3D) cell cultures, in use for over a century, have been instrumental in biomedical research. Nonetheless, they are unable to replicate the intricate spatial and chemical dynamics of living tissues or to predict drug responses accurately. There are ongoing endeavors to narrow this disparity between in vitro and in vivo models. One such initiative originally stems from the realms of microelectronics and micro-electro-mechanical systems.1 Leveraging technology from these fields, it enables the precise manipulation of fluids within submillimeter closed structures such as channels and chambers,2 earning it the designation microfluidics (MFs). MF culture devices are also called organs-on-chips, in referral to their origin in the field that normally manufactures computer microchips.3 In these devices, single or multiple cell types as well as tissue fragments can be cultured. With the precise adjustment of flow, control over parameters such as nutrient levels and gradients and the removal of metabolites is gained.4,5 These benefits enable high cell concentrations in culture, promoting more cellular cross-talk and paracrine communication.6 Especially for prolonged culture periods, MF technology has demonstrated enhanced tissue viability compared with conventional static systems, which do not employ continuous media exchange.7,8

Many patients with childhood cancers or specific metabolic, hematological (e.g., sickle cells disease and thalassemia major), and immunologic diseases are exposed to gonadotoxic high-dose chemo- and radiotherapy, with the risk of long-term infertility, often before a hematological stem cell transplantation.9,10,11 Due to a 5-year survival rate of 80% and more for childhood cancer, many patients are reaching reproductive age, making infertility an increasingly consequential side effect.12,13,14 In contrast to adult patients, prepubertal patients undergoing gonadotoxic treatment cannot rely on the cryopreservation of spermatozoa for fertility protection, since sperm production only starts at puberty. To allow these patients to father their own biological children in adulthood, new methods for human in vitro spermatogenesis or in vitro propagation of human spermatogonial stem cells (SSCs) need to be developed. This is particularly important for cancer patients. Autotransplantation of testicular tissue containing SSCs, a method that has already led to living offspring in nonprimates, is not a viable option for that patient group because of the risk of cancer cells being reintroduced with the transplant.15,16 According to the current guidelines, even though there are no clinically established uses, cryopreservation of testicular biopsies may be proposed to high-risk patients before gonadotoxic treatment in the hope of successful therapy options in the future.17,18 MF could be utilized to achieve such progress.

For many years, sperm counts in the male population are reported to have declined.19,20 Because of the significant implications for public health and society, research on this is urgently needed. While sperm count reduction has been associated with many lifestyle and environmental influences, the molecular mechanisms remain to be understood.21,22,23,24 The current toxicological testing, which includes not only environmental factors but also essential parts of the drug approval process, largely depends on animal testing. However, in vitro models are also encouraged in accordance with the replace, reduce, and refine (3Rs) principle.25,26 MFs are a potentially powerful tool for the enhancement of animal-free and time-efficient models to reduce animal experiments.

TECHNICAL CONSTRUCTION OF MF DEVICES (MFDS) USED IN TESTICULAR CULTURE

Materials

To be suitable for the production of MFDs, materials require specific properties that permit culture. Only few materials have an appropriate combination of being low-cost, easy to work with as well as gas permeable. The most commonly used in current MF setups is polydimethylsiloxane (PDMS), a silicon polymer, that is optically transparent. This material cannot be considered optimal, primarily because of its adsorption of hydrophobic molecules, which can complicate experiments where precise concentrations of these molecules are crucial.27,28,29 PDMS also releases uncross-linked oligomers that may damage cells when incorporated into their membranes. Other drawbacks include the tendency to revert to a hydrophobic state following certain surface treatments and a different refractive index from that of glass, which complicates the use of high-resolution microscopy.30,31,32

Alternatives to PDMS include thermoplastics, such as polymethylmethacrylate (PMMA), polystyrene (PS), polycarbonate (PC), and cyclic olefin copolymer (COC), as well as glass. Apart from glass, PC and COC have been shown to adsorb close to no hydrophobic molecules after treatment. Even though inferior to PDMS in gas permeability and biocompatibility, they have superior optical clarity and chemical resistance, making them the better choice in specific cases.33

Ultimately, many different materials can be used for the construction of testis-on-chip (ToC) devices depending on the requirements of the specific study.

In this review, among the eleven ToC devices examined, four employed PDMS for both the top and bottom layers of the device,34,35,36,37 five utilized a PDMS layer bonded to a glass slide,38,39,40,41,42 one used a PDMS slide bonded to a PDMS-coated glass slide,43,44 and one incorporated both PDMS and PMMA layers.45 Yamanaka et al.42 explained their choice for glass as the second layer because of superior optical qualities. This could be similarly assumed for Sharma et al.41 as well as Kanbar et al.,39 who used live imaging during their experiments. Finally, the choice of material depends on a combination of cost, intended use, equipment available, and the expertise of the user.

Fabrication techniques

Most commonly, MFDs are produced by photolithography to create a master mold. This is a well-established method capable of producing features down to 100 nm, establishing it as the standard for high-resolution applications, and is therefore frequently used in the production of the ToC devices.35,36,37,38,39 42,43 The master mold serves as a negative template and is employed in soft lithography to cast the final device. The final construction can be capped with a glass slide or a second PDMS-block using plasma oxidation methods or by applying pressure to form noncovalent bonds.46

In some cases, other techniques are used because of the accessibility of equipment, cost, or time required. The following have been reported in the testicular MFDs mentioned here.

Sharma et al.41 employed a practical yet less precise method using a stereolithography (SLA) printer with a print precision of ±50 µm. While their MFD, therefore, theoretically allowed features ranging from 75 µm to 175 µm for an intended size of 125 µm, microscopy-based validation revealed actual size differences closer to approximately 5 µm.

Similarly, AbuMadighem et al.34 utilized a PolyJet printer with a stated precision of ±100 µm for 100 µm features. However, lacking validation or imaging data, the actual deviations in the fabricated device remain uncertain. Given that the primary function of these small features was to contain a gel, and no gel leakage was reported, such size variability may be inconsequential.

Yet, Naeemi et al.45 employed a cutter plotter with a resolution of 25 µm, although no notable features were produced at this scale. This group also utilized a micromilling machine with a 2 µm-resolution to fabricate a MF channel on PMMA.

Önen et al.40 used deep-reactive ion-etching to produce their master mold, a method allowing high-accuracy manufacturing.47

Chambers and limiting walls

To produce a functional ToC platform, the sample/cells need to be immobilized in the medium flow. This can be achieved in a number of ways. While most setups have solely used chamber-based trapping methods, alternative strategies include hydrodynamic trapping,48,49 immobilization within individual hanging drops,50 cell adhesion by surface patterning of the MFD with biofunctional molecules (e.g., cell antibodies or collagen),37,43,51 as well as cell adhesion through noncovalent interactions.52

Chamber-based MF setups must permit medium access through the chamber’s containing structures. To achieve this, these need to be permeable, which can be accomplished by employing a porous membrane (Figure 1a),35,36,38,45 arrays of micropillars (Figure 1b),37,39,40,41,42,43 or permeable gels such as agar (Figure 1c).34 Depending on the size of the pores, the nutrients may only pass through by diffusion or convection. Two ToC studies investigated the precise shear forces the tissues experienced within the chambers to determine whether the tissue was subjected to stress levels exceeding physiological thresholds.40,41

Figure 1.

Illustrative layouts of three distinct microfluidic (MF) setup categories, highlighting key technical details from the respective studies. (a) Membrane systems, (b) micropillar systems, and (c) gel-based system. Green arrows (in a and b) indicate the direction of pump-driven flow of the culture medium. The gel-based system in c does not include a pump-driven media flow (no green arrows). In a, the membrane (yellow) on top of the tissue chamber (pink) reduces the influence of the damaging shear forces on the cultivated tissue. Cultivation medium enters the tissue chamber through the membrane pores (thin curved green arrows). In b, the cultured tissue (pink) is located in the center of a battery of micropillars, intended to prevent/minimize the influence of shear forces on the tissue. Cultivation medium reaches the cultivated tissue through the distances between the micropillars. In c, green symbolizes the culture medium, flowing into the outer channels (green) by gravity and hydrostatic pressure (red arrows), where the medium is held. The yellow chambers contain the agar as the retaining gel. Cells, mixed with methylcellulose gel, are located in the central tissue chamber (pink). Cultivation medium passes from the outer channels (green) through the agar chamber (yellow) to the centrally localized cell chamber (pink) by diffusion (red arrows between the colored chambers). Tables below the schematic illustrations provide the individual technical details for the systems of each respective setup category. NA: not available.

For chamber dimensions, Komeya et al.35 investigated the maximum tissue thickness capable of adequately supplying nutrients to the central regions through diffusion. They identified this as being between 300 µm and 400 µm (Figure 2a), theoretically allowing for tissue thicknesses of up to 800 µm. A few setups came close to this thickness, but none have reported poor tissue viability directly linked to insufficient nutrient diffusion. Three studies used tissue chambers with a maximum diameter of 800 µm.39,41,42 While Kanbar et al.39 as well as Sharma et al.41 observed regions of poor core integrity or necrosis, the localizations were not directly linkable to diffusion distance. Yamanaka et al.42 associated the poor spermatogenesis in the central region of their tissues to cramped space, as this issue did not arise when there was adequate room for growth (Figure 2b and 2c). Önen et al.40 used a chamber with a maximum diffusion distance of 325 µm (diameter: 750 µm) and reported neither necrosis nor compromised tissue integrity.

Figure 2.

Limit of nutrient diffusion in cultured testicular tissue and the limitations in tissue preservation due to space restriction. (a) Molecular diffusion in neonatal mouse testis tissue. Testicular tissue was cultured in a slit device, and photos were taken on days 13, 25, and 44. The region of the tissue distant from the medium flow channel became necrotic owing to a shortage of nutrients. Oxygen was supplied evenly through the ceiling made of PDMS. The distance of nutrient diffusion from the medium flow through the testis tissue to maintain it was about 400 µm at most in this experiment. Scale bar=400 µm. Reproduced from Komeya et al.,35 under a Creative Commons CC BY license. (b) When a large neonatal mouse testis tissue was loaded, its growth is limited by the pillars, leaving the central region compressed and GFP-negative. Reprinted from Yamanaka et al.42 (copyright: 2018), with permission from Elsevier (license number: 5878730823096, license date: Sep 30, 2024). (c) A small piece of testis tissue, taken from a 3.5 days postpartum (dpp) mouse, loaded in the middle of the tissue chamber. It showed progressive enlargement and Acr-GFP expression. Reprinted from Yamanaka et al.42 (copyright: 2018), with permission from Elsevier (license number: 5878730823096, license date: Sep 30, 2024). PDMS: polydimethylsiloxane; Acr: acrosin; GFP: green fluorescent protein.

Pumping mechanisms

Various pumping mechanisms are possible for MFDs. Most are external pumps, such as the syringe pump used for the first ToC from Komeya et al.35 as well as the syringe pump employed by Kanbar et al.39 Other systems included on-chip gravity-driven devices,36,40,42 on-chip peristaltic pumps,38 and a sample injector for perfusion.37 One group,34 although claiming to have created an MFD, did not expose their system to flow (Figure 1c), but since the group aims to use this same device for perfusion experiments in the future, it is included in this review.

For Komeya et al.,35,36 the syringe pump posed constraints owing to its occupation of incubator space which limited the number of MFDs attachable; a pumpless, gravity-driven perfusion system was developed by integrating a resistive channel into the MFD, enabling a more stable flow rate. Their experiments, with flow rates varying from 0.05 µl min⁻¹ to approximately 2.0 µl min⁻¹, showed in vitro spermatogenesis to be as effective as with constant external perfusion. The flow rates of all MFDs, discussed here, are summarized in Figure 1.

Baert et al.38 used an on-chip peristaltic pump providing a pulsatile flow averaging about 9.3 µl min⁻¹. Developed by Wu et al.53 in 2008, it requires pressure being applied successively to three PDMS membranes to enable flow without large external medium reservoirs. Baert et al.38 used this setup to achieve physiological extracellular-to-intracellular fluid volume ratios in their MF setups. In the human body, the proportion of extracellular fluid in relation to tissue is 20%,54 so approaching this value might improve outcomes. Byun et al.55 provided an in-depth review on pumping mechanisms.

ADVANCING THE RECREATION OF SPERMATOGENESIS VIA MFS

Cell sources and tissue engineering

The history of testicular in vitro culture began with the culture of intact testicular fragments, exploiting the native scaffolding and cellular interactions of the tissue, which cannot be achieved in 2D systems, nor in current 3D systems.56,57,58,59 With advancements in in vitro culture techniques and biotechnology, researchers have tried many 2D and 3D setups to replicate specific testicular functions, most prominently spermatogenesis.60

As with the first static in vitro testicular culture, the first MF testicular culture was also of intact testicular tissue.35 Building on success with murine static culture, in which complete spermatogenesis was recreated in vitro, the Ogawa group from Yokohama City University (Yokohama, Japan) established a simple setup with medium perfusing a chamber with immature testicular tissue (ITT) from mice. By introducing flow, they were able to induce complete spermatogenesis at higher rates and for longer than that in their static air–liquid interphase experiments.35,61 Following the successful generation of spermatozoa and the production of live mouse offspring, this research group maintained the same experimental configuration in two subsequent studies.35,36,42 While these largely retained the methodology of their initial work,35 they incorporated improvements to facilitate handling and enhance evaluation. Specifically, the inclusion of an on-chip pump facilitated easier handling, and the use of a glass slide bottom allowed for high-resolution microscopy, improving the quality of evaluation (see “Materials”). In a subsequent study, the authors noticed reduced spermatogenesis when they used large ITT fragments that did not leave room for growth of the tissue (see “Chambers and limiting walls”).42

A similar setup with murine ITT was published by Önen et al.40 in 2023, achieving spermatogenesis up to round spermatids. With this, the group compared a medium conditioned by culture with bone marrow-derived mesenchymal stem cells (BMSCs) with an unconditioned medium (further discussed in “Medium”).

Kanbar et al.39 used porcine ITT in a system that otherwise strongly resembled the initial system of Komeya et al.35 They used pigs because of their closer resemblance to human spermatogenesis than that of mice or rats.62,63 They evaluated the experimental results with the MF setup and ITT from approximately 5- to 9-day-old piglets alongside three static setups (culture insert with a polytetrafluoroethylene [PTFE] membrane, agarose without a PDMS component, and agarose covered by PDMS chip).39 While spermatogenesis was successfully initiated in all four systems, investigated with the markers ubiquitin C-terminal hydrolase L1 (UCHL1; PGP9.5), DEAD-box helicase 4 (VASA), synaptonemal complex protein 3 (SYCP3), CAMP responsive element modulator (CREM) (germ cells), SRY-box transcription factor 9 (SOX9), insulin-like 3 (INSL3; somatic cells), and marker of proliferation Ki-67 (MKI67; proliferating cells) as well as the hormone testosterone (T) enzyme-linked immunosorbent assay (ELISA), only one of the static setups (agarose with a PDMS cover chip) outperformed the control (culture with PTFE membrane) by a slightly higher efficiency of germ cell differentiation. Since this study showed little difference in efficiency between the culture systems, Kanbar et al.39 postulated that the current limiting factor on pig spermatogenesis is adequate media composition (e.g., growth factors, dosage, and timing of administration).

Sharma et al.41 employed primate ITT from marmosets and testicular tissue from adult patients with gender dysphoria in a comparable experimental setup. Their primary objective was to establish the device rather than investigate the progression of spermatogenesis achieved. However, they did compare the MF system of both primate species against static controls using membrane-based culture dishes. Significant findings from their experiments with the human testis included the persistent integrity of the basement membrane (for at least 5 days), superior cell survival, the presence of peritubular cells, somatic Sertoli cells, and cohorts of differentiating spermatocytes and spermatids. With the MF setup, they were able to maintain culture for up to 11 days. The marmoset ITTs were utilized to explore the response to hormonal stimulation (further discussed in “Medium”). The authors have now set out to use this established model to screen for suitable culture parameters to achieve successful primate spermatogenesis.

Naeemi et al.45 designed testicular organoids through culture of isolated murine SSCs from Naval Medical Research Institute (NMRI) mice (age of 7 days postpartum [dpp]) on a mix of decellularized testicular tissue (DTT) and hyaluronic acid. DTT was first used for in vitro models of the human testis in 2017 by Baert et al.64 and many groups have subsequently used DTT as a structural scaffold with testis-specific extracellular matrix (ECM) proteins to provide a microenvironment as close as possible to in vivo conditions.65,66,67 The addition of hyaluronic acid to ECM is probably to replace the scaffold with the ECM secreted by the seeded cells.68 For the cultivation of adipose-derived stem cells, this combination has shown great promise.69 The organoids generated by Naeemi et al.45 were able to support mouse in vitro spermatogenesis at least to step 11 of spermiogenesis (elongating spermatids), which was proven through immunohistochemistry with the markers promyelocytic leukemia zinc finger protein (PLZF), tektin 1 (TEKT1), and transition protein 1 (TP1).70

AbuMadighem et al.34 induced on-chip spheroid formation by seeding testicular cells, isolated from neonatal male mice (7–8 dpp, CD-1® mice), in methylcellulose hydrogel. This approach succeeded in inducing mouse SSCs up to the round spermatid stage of spermiogenesis. Their control, a well plate system, also achieved this stage but had a significantly lower percentage of late-stage/postmeiotic cells (CREM⁺ or Acrosin [ACR]⁺). Higher numbers of haploid cells were found in the MF as well as better preserved viability and more mature Leydig cells. Aspects of testicular microscopic architecture were recreated in the MF system, such as Sertoli cells extending branches throughout the spheroids, thereby giving structural support as in vivo.

Despite reports indicating that a relatively high cultivation temperature (37°C) is disadvantageous for spermatogenesis,71 three groups using mouse MF testicular tissue culture34,40,43 and two groups using human MF testicular tissue culture37,38 employed it in their experiments, whereas the other groups used lower temperatures of 35°C or 34°C.35,36,39,41,42,45 The reason for choosing 37°C in the two human studies37,38 could be based on the fact that the cocultured cells from the kidney or liver grow better at this temperature, as both systems were multiorgan systems.

We give an overview of the studies on testicular MF culture to date with the different sample types used (Figure 3).

Figure 3.

The different sample types employed for testicular microfluidic (MF) culture.

Medium

Most groups based their choice of medium on previous work and experience and did not compare different media in their MF setup. Nevertheless, we contextualize here the different components used in MF testicular culture from research published on their effects on in vitro spermatogenesis.

For MF testicular devices, different basal media have been used, most being variations of Eagle’s essential medium (Dulbecco’s minimum essential medium [MEM], α-minimum essential medium [α-MEM], α-MEM/F12, and DMEM/F-12 nutrient mixture [DMEM/F12]). While these variants are all based on basal medium, this does not necessarily speak to an identical capacity of each medium to induce spermatogenesis. Additional vitamins, amino acids, and/or other nutrients have been added to the variants providing an adequate environment depending on the need of the cultured cell type.61,72 AbuMadighem et al.34 used a different medium base rarely used for spermatogenesis.73,74 To identify the best basic medium for murine in vitro spermatogenesis, Sato et al.61 compared Roswell Park Memorial Institute (RPMI) 1640, Dulbecco’s modified eagle’s medium (DMEM), DMEM/F12, and α-MEM (+10% fetal bovine serum [FBS] each), and found RPMI and α-MEM to be most suitable. Early in their research on spermatogenesis, the group around Takehiko Ogawa from Yokohama City University identified RPMI with AlbuMax (40 mg ml⁻¹) as very efficient in inducing in vitro spermatogenesis in mouse testicular tissue.61 These scientists compared DMEM, DMEM/F12, α-MEM, and RPMI and decided on utilizing RPMI or α-MEM for testicular culture. Their use of AlbuMax resulted from the quest for a serum replacement, since they then believed that FBS contained spermatogenesis-inhibiting factors (a concept later disproven). AlbuMax was deemed to be the critical component of knockout serum replacement (KSR), the most proficient replacement promoting spermatogenesis, so the group also used this in their MF setups.35,36,42

FBS is the most widely used growth supplement for cell culture media due to its high content of embryonic growth-promoting factors.75,76 Because of its position as a standard for animal cell culture, it has also been used in four of the reported MF setups.37,40,43,45 Before starting microfluidic studies using testicular tissue in general, Sato et al.61 sought to refine medium composition for murine spermatogenesis, suspecting FBS to contain factors inhibiting spermatogenesis. Since FBS is isolated from animals, the composition is batch-dependent and unknown to their users.76 Although it was later disproven that FBS inhibits murine spermatogenesis, the Ogawa group found KSR more capable at inducing spermatogenesis.61 They attributed this to a particular component of KSR, the AlbuMax (lipid-rich albumin),61 which is efficacious at a 10% concentration in supporting in vitro spermatogenesis in different species.64,77,78,79 Most of the MF studies discussed here therefore included KSR to their basal medium.34,35,36,38,39,40

In 2021, Matsumara et al.80 found that the effect of AlbuMax (40 mg ml⁻¹) was specific to mice and hardly induced the expression of a meiotic germ cell marker in rats. This is not particularly surprising, since even in a different mice strain, the success of Sato et al.61 in 2011 (with KSR as supplement) could not be replicated.81

A particularly interesting medium was used by Önen et al.40 They investigated whether secretions of BMSCs, a cell type of similar embryonic origin, differentiation, and immunomodulatory capacity as Sertoli cells,82,83 could improve spermatogenic progress in vitro as well as neonatal mouse SSC maintenance and propagation.40 The group thus applied a medium proven suitable for 24 h-BMSC culture as a base for the testicular culture medium. In comparison with untreated medium, higher numbers of differentiating spermatogonia (KIT proto-oncogene, receptor tyrosine kinase [c-KIT]-positive cells) and total germ cells (VASA-positive cells) were reported.40 Furthermore, the PLZF-positive and the spalt-like transcription factor 4 (SALL4)-positive SSC/progenitor cell population could be maintained for 42 days. This result supports the findings of a similar study the group had performed on static culture.84

AbuMadighem et al.34 investigated the effects of retinoic acid (RA) and T in their MF culture by preculturing spheroids formed from single cells for 5 weeks before 2 additional weeks of culture with RA or T. Effects were evaluated by immunostaining as well as RNA expression analysis for the markers PLZF, cadherin 1 (CDH1), c-KIT (spermatogonia), VASA (total germ cells), CREM, boule homolog, RNA-binding protein (BOULE; meiotic cells), ACR, protamine 1 (PRM1; postmeiotic cells), vimentin (VIM), and alpha-smooth muscle actin (α-SMA; somatic cells). While T only increased expression of one marker for Sertoli cell function (androgen receptor, AR), RA elevated the expression of the premeiotic markers PLZF and c-KIT as well as multiple markers for Sertoli cell activity (transferrin [TF], follicle-stimulating hormone receptor [FSHR], and AR). Naeemi et al.45 also included RA into their medium for MF testicular cell culture, but not on the basis of results from MF experiments, but from benefits shown in static in vitro culture as well as in vivo.85,86

Sharma et al.41 investigated the response to hormonal stimulation in their experiments with marmoset ITT. Strong hormonal stimulation with follicle-stimulating hormone (FSH), human chorionic gonadotropin (hCG), and marmoset serum resulted in a rise of T and estrogen secretion and in the maturation of Leydig cells, less apoptosis, more organized epithelium, and better-preserved structural integrity. Whereas the trend was homogeneous in the nine stimulated marmoset ITTs, the degree of response varied significantly, even though all ITTs were isolated from the same animal.

MF IN VITRO CULTURE TO MODEL REPRODUCTIVE TOXICITY

To date, three MF models for reprotoxicity have been reported, two of coculture with important organs for the metabolism of toxic substances, the liver, and the kidneys.37,38 Dong et al.43 investigated the impact of titanium dioxide (TiO₂) on a biomimetic blood–testis barrier.

A proof of concept regarding liver-dependent toxicity to the male gonads was created by Baert et al.38 by establishing a coculture of liver spheroids with testicular organoids, generated by placing human adult primary testicular cells on an agarose support and allowing them to form organoids over 3 days, before starting the MF culture. All primary testicular cell types (Sertoli, Leydig, peritubular myoid cells, and germ cells) were detected in the cultured testicular organoids. Liver spheroids were formed from differentiated HepaRG cells87 and primary hepatic stellate cells by culturing them for 3 days in a liver-specific medium. The liver-specific medium had previously been identified as the most suitable medium for the coculture on the basis of experiments evaluating metabolic activity and cell damage after the cultivation in the different media (liver-specific versus testis-specific versus combined medium). The ability of the model to demonstrate reproductive toxicity was examined in experiments with cyclophosphamide, a prodrug, presenting toxic effects only if metabolized to 4-hydroxycyclophosphamide by cytochrome P450 enzymes, a mainly hepatic process. The coculture experiments supplemented with cyclophosphamide revealed a marked loss of germ cells, but as cell viability was not reduced, the drug specifically targeted rapidly proliferating cells.38

The kidney–testis MFD by Xiao et al.37 was a 2D coculture, connected via endothelium-covered chambers, coated with collagen type I for improved adherence. The testicular cells used were immortalized cells from a nonseminomatous malignant germ cell tumor (NTERA-2 cl.D1). Endothelium and kidney were imitated by human umbilical vein endothelial cells (HUVEC) and tubular epithelial cells (human kidney 2 [HK-2]), respectively. The MFD was developed to test toxic effects of microplastics (MPs), defined as 0.5–5000 µm in size, and nanoplastics (NPs), which are even smaller. Such plastic particles are the result of natural chemical and biological degradation of waste plastic or are intentionally produced, e.g., for the use in waterproof coatings.88,89 Humans are exposed to MPs/NPs via inhalation, ingestion, and skin contact, and with increased production and disposal of plastic products and low biodegradation rates, exposure is projected to rise.90,91 Owing to their hydrophobic surface, not only their own toxicity but also their ability to absorb other toxic substances and pathogenic microorganisms pose health concerns.92,93 In exposure experiments, the group around Xiao et al.37 showed that MPs/NPs are internalized by the cells via endocytosis, that cell viability is reduced and that cancer-related signaling pathways are upregulated. The group around Dong et al.43 continued efforts to develop functional MF models for reproductive toxicity by creating a biomimetic blood–testis barrier. For this, murine Sertoli, spermatogonial, and endothelial cell lines (TM4, GC-1, and C166, respectively) were cultured in interconnected chambers designed to mimic the structure of seminiferous tubules. The model’s functionality was assessed by immunofluorescence staining of tight junction proteins in Sertoli cells, determining the barrier permeability to fluorescein isothiocyanate (FITC)-dextran and measuring glucose levels in the supernatant, which indicate barrier integrity and system stability, respectively.43 The model was then exposed to TiO₂ NPs,43 a group of metal oxide NPs commonly used in consumer products for their biocidal, photocatalytic, and whitening properties.94,95,96 Numerous in vivo studies have shown the toxic effects of TiO₂ on male gonads, but the complex physiological processes of animal models make it challenging to study cellular interactions within barriers at the molecular and cellular levels.97,98 After exposure to TiO₂ NPs (100 µg ml⁻¹), gene expression and protein levels in Sertoli and germ cells were examined. In Sertoli cells, the expression of tight junction proteins, as well as enzymes involved in lactate production, crucial for supplying energy to germ cells, was reduced. In germ cells, cytokine signaling was reduced, and the production of reactive oxygen species increased, potentially leading to an imbalance between oxidative stress and antioxidant defense systems, which may impair spermatogenesis.43

CONCLUSION

MF culture represents a promising tool for both in vitro spermatogenesis and reprotoxicity studies. Since a functioning testicular microenvironment is essential for its success, further improvement should include assessing markers that reflect testicular tissue integrity (e.g., laminin subunit alpha 1 [LAMA1] expression or immune reactions).9,99,100 While testicular MF culture is still in its infancy, and while there is evidence pointing to its benefit for spermatogenesis in vitro, many aspects of testicular culture conditions (e.g., oxygen and growth factor concentrations, scaffolding, and ECM components) need to be improved before in vitro spermatogenesis can become clinically applicable. Furthermore, since the methodology for both the in vitro spermatogenesis and the reprotoxicity models is diverse, with different species and sample types being used, comparability of the approaches is limited. Consequently, culture conditions in the future should be optimized in a species-specific and sample type-dependent manner to identify the conditions necessary to induce in vitro spermatogenesis.

Table 1 provides an overall summary of the eleven MF studies described in this review, including their main objectives and key findings.

Table 1.

Summary of the research conducted with testicular cells and microfluidics

Study type	Reference	Cell source	Aim	Main result	Methodologies	Markers
In vitro spermatogenesis study	Komeya et al.35 2016*	Neonatal mice (0.5–5.5 dpp)	Imitation of microvascular flow for long-term organotypic culture	T production and mouse spermatogenesis for 6 months; functional sperm for healthy offspring after micro-insemination	IF ECLIA LC-MS/MS ICSI, ROSI	GFP PNA SCP3 T SPT fertility
	Komeya et al.36 2017*	Neonatal mice (0.5–4.5 dpp)	Pumpless MF system for slow medium flow	3-month culture with induction and maintenance of mouse in vitro spermatogenesis	Microscopy/live imaging IF	GFP EdU GFRA1 TRA98
	Yamanaka et al.42 2018*	Neonatal mice (0.5–5.5 dpp)	Test of simpler designed and easier-to-produce MF system	Induction and maintenance of mouse in vitro spermatogenesis; way of sample introduction into MF device during its production	Microscopy/live imaging	GFP
	Önen et al.40 2023	Neonatal mice (6 dpp)	Evaluation of efficacy of new pumpless ToC platform	SALL4+ and PLZF+ SSC maintenance; increase of differentiating SPG, total germ cells, meiotic cells, T production for 42 days (compared to hanging drop and without BMSCs)	IHC FACS LC-MS/MS 3D COMSOL Multiphysics®	SALL4 c-KIT PLZF VASA T Shear stress
	Naeemi et al.45 2023	Neonatal mice (7 dpp)	Design/test of hydrostatic pressure-based MF system	Expansion of SSCs	Microscopy IHC	Acridine orange PLZF TEKT1 TP1
	AbuMadighem et al.34 2022	Neonatal mice (7–8 dpp)	Test of new ToC platform for in vitro spermatogenesis	Single cells formed spheroids; 95% cell viability after 7 weeks; in vitro spermatogenesis up to and including meiosis II	IFa/real-time PCRb FACS Trypan blue staining	ABPb ACRa,b ARb BOULEa,b c-KITa,b CDH1a,b CREMa,b FSHRb GAPDHb PLZFa,b PRM1b SALL4b TFb SMAa VASAa,b VIMa Ploidy state of cells Cell viability
	Kanbar et al.39 2022	Prepubertal pig	Comparison of static and dynamic (MF) conditions	Best germ cell survival and differentiation in the static set-up with modified conditions (agarose gel and PDMS chamber)	IF/IHC ELISA Live imaging	CASP3 CREM INSL3 Ki-67 MDA SOX9 SYCP3 UCHL1 VASA T
	Sharma et al.41 2022	Prepubertal marmoset (6 months) and adult human (gender dysphoria patients after sex reassignment surgery)	Test of ToC platform in ex vivo culture of primate testis tissue to study in vitro spermatogenesis and endocrine dynamics	Marmoset samples: viable cell populations after 9 days; T and estradiol production after gonadotropin stimulation; improved tissue maintenance under stimulatory conditions Human samples: viable cell populations after 11 days	ELISA (marmoset samples) Live-dead staining Live imaging 3D COMSOL Multiphysics®	T Estradiol Cell viability Shear stress
Reprotoxicity study	Baert et al.38 2020	Human adult testis, liver	1st step for reprotoxicity model	Upregulation of cytochromes in liver spheroids and germ cell loss after addition of cyclophosphamide	Photometry IFa/real-time PCRb ELISA	Glucose Lactate LDH ALBa,b CYP1A1b CYP2A6b CYP2B6b CYP2C9b CYP2C19b CYP3A4a,b DDX4a Ki-67a
						MDR1b MRP1b SMAa SOX9a STARa TUNELa UCHL1a UGT1A1b UGT2B28b Inhibin B T
	Xiao et al.37 2023	Human testis (NTERA-2 cl.D1), kidney	To study the toxicity of NPs	NP entrance into kidney and testis cells via endocytosis; activation of cancer-related signaling pathways	Glucometry IF ELISA Cell counting (KIT CCK-8)	Glucose CLDN2 TNF-α AKT BAD ERK JNK1/2/3 MDM2 NRF2 PI3K P38 P53 RAS RTK TNF-α-R VEGF Cell viability
	Dong et al.43 2024	Mouse testis (Sertoli cell line TM4, SPG cell line GC-1, and endothelial cell line C166)	Study of spermatogenesis and BTB-damaging effects of TiO2 nanoparticles and elucidation of the mechanisms behind	TM4 cells: spermatogenesis energy-substrate supply reduction (glucose metabolism, especially lactate production including key proteins and metabolites) GC-1 cells: disturbance of cell proliferation-regulating pathways and interference in glutathione metabolism	ELISA/photometry IF WB RNA-sequencing	Glucose Glutathione Lactate ROS CLDN11 CTNNB1 ZO1 ACTB CXCL13 GPX4 GSR HK1 LDH PFKM p-STAT3 STAT3 Transcriptomic and metabonomic markers

*A study from the Ogawa group; ^aIF; ^breal-time PCR. ABP: androgen-binding protein; ACR: acrosin; ACTB: actin beta; AKT: AKT serine/threonine kinase; ALB: albumin; BAD: BCL2-associated agonist of cell death; BMSCs: bone marrow-derived mesenchymal stem cells; BOULE: boule homolog, RNA-binding protein; BTB: blood–testis barrier; CASP3: caspase 3; CLDN2: claudin 2; CLDN11: claudin 11; CREM: CAMP responsive element modulator; CDH1: cadherin 1; CTNNB1: catenin beta1; c-KIT: KIT proto-oncogene, receptor tyrosine kinase; dpp: days postpartum; CXCL13: chemokine 13; CYP1A1: cytochrome 1A1; CYP2A6: cytochrome 2A6; CYP2B6: cytochrome 2B6; CYP2C9: cytochrome 2C9; CYP2C19: cytochrome 2C19; CYP34A: cytochrome 3A4; ECLIA: electro-chemiluminescence immunoassay; EdU: 5-ethynyl-2′-deoxyuridine labeling; ELISA: enzyme-linked immunosorbent assay; ERK (MAPK1): mitogen-activated protein kinase 1; FACS: fluorescence-activated cell sorting; FSHR: follicle-stimulating hormone receptor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GFRA1: GDNF family receptor alpha 1; GPX4: glutathione peroxidase 4; GSR: glutathione reductase; HK1: hexokinase 1; ICSI: intracytoplasmic sperm injection; IF: immunofluorescence staining; IHC: immunohistochemistry; JNK1/2/3: JUN N-terminal kinase 1/2/3; Ki-67 (MKI67): marker of proliferation Ki-67; LC-MS/MS: liquid chromatography-tandem mass spectrometry; LDH: lactate dehydrogenase; MDA: malondialdehyde (marker for oxidative stress/lipid peroxidation); MDM2: MDM2 proto-oncogene; MDR1 (ABCB1): ATP-binding cassette subfamily B member 1; MF: microfluidic; MRP1 (ABCC1): ATP-binding cassette subfamily C member 1 (ABCC1 blood group); NPs: nanoplastics; NRF2 (NFE2L2): NFE2-like BZIP transcription factor 2; NTERA-2 cl.D1: human testis cancer cell line; P38: P38 kinase; P53: P53 kinase; PDMS: polydimethylsiloxane; PFKM: phosphofructokinase, muscle; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; PLZF: promyelocytic leukemia zinc finger protein; PNA: peanut agglutinin; PRM1: protamine 1; RAS: rat sarcoma viral oncogene; ROS: reactive oxygen species; ROSI: round spermatid injection; RTK: receptor tyrosine kinase; SALL4: spalt-like transcription factor 4; SCP3: synaptonemal complex protein 3; SMA: alpha smooth muscle actin; SOX9: sry-box transcription factor 9; SPG: spermatogonia; SSC/SSCs: spermatogonial stem cell(s); STAR: steroidogenic acute regulatory protein; STAT3: signal transducer and activator of transcription 3; p-STAT3: signal transducer and activator of transcription 3, phosphorylated; SPT: spermatid; T: testosterone; TEKT1: tektin 1; TF: transferrin; TiO₂: titanium dioxide; TNF-α: tumor necrosis factor-alpha; TNF-α-R: tumor necrosis factor-alpha-receptor; ToC: testis-on-chip; TP1: transition protein 1; TRA98 (GCNA): germ cell nuclear acidic peptidase; TUNEL: TdT-mediated dUTP nick-end labeling; UCHL1: ubiquitin C-terminal hydrolase L1; UGT1A1: UDP glucuronosyltransferase family 1 member A1; UGT2B28: UDP glucuronosyltransferase family 2 member B28; VASA (DDX4): DEAD box helicase 4; VEGF: vascular endothelial growth factor; VIM: vimentin; WB: western blot; ZO1: zonula occludens protein 1

AUTHOR CONTRIBUTIONS

BMS and KvK conceptualized this work and wrote the manuscript. BMS, KvK, and HIH carried out the literature research and created the figures and tables. KvK supervised the project. AS organized the financial resources. All authors reviewed and edited the manuscript, contributed intellectually to the content of the manuscript, and read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.