Bio-electrosprayed bovine sperm remain viable and fertilize oocytes

Bio-electrospraying,¹ the technology of electrospraying living cells, has undergone rigorous development since 2005. From then, the technology has been exposed to over 600 cell types ranging from mammalian to non-mammalian cells, to stem cells and whole fertilized embryos.² The technology works on the principle of applying a high voltage to a conducting needle accommodating the flow of a suspension containing living cells. Centrally below, (usually at a distance of 10 mm) away from the charged needle exit, lays a ring grounded electrode. The electric field between the charged needle and the grounded electrode accelerates the charged cell suspension exiting the needle toward the grounded electrode. This process results in the formation of either a three-dimensional (3D) conical spray plume or a stream of droplets (which could be pulsed for generating a drop on demand) for deposition. The technology is governed by several parameters, namely, the applied voltage to the needle, the flow rate of the cell suspension to the needle, the suspension properties including electrical conductivity, viscosity, density, relative permittivity, and surface tension, and the equipment set-up such as needle geometry and diameter, the distance between the needle and the grounded electrode, and electrode geometry. When the right balance is obtained between all these operational parameters, a stable liquid cone and jet is seen to form at the needle exit, also referred to as the cone-jet mode, resulting in either a stable continuous spray or a stream of a near-monodispersed distribution of droplets. During stable conditions, the process could also be pulsed; notably, the uniqueness of this jetting technology over classical jetting technologies such as 3D printing, is the ability of bio-electrosprays/electrospray to handle large volumes of materials (such as multi-materials/material gradients) in suspension. The larger volumes of materials are handled through large inner bore needles (>1000 μm), while remaining capable of generating residues from a few nanometers to tens of micrometers.³ Note when handling nanomaterials/biomolecules, the generated residues could be in the nanometers, and when handling micromaterials/living cells, the residues are usually in the tens/hundreds of micrometers.³ In both scenarios, the loading/density (together with the material dimensions) of the prepared suspension has a significant effect on the generated residues. Interestingly, the capacity of electrosprays to handle high volumes of multi-materials/material gradients as suspensions, while avoiding needle blockage, is achieved, as the process avoids the Barus effect taking form at the needle exit. This effect, unlike in the case of electrosprays/bio-electrosprays, is prevalent in all 3D printing/bioprinting manifestations. The Barus effect,⁴ along with many other characteristics, is well-known to encourage the negative effects on printing resolution and residue integrity to increasing needle blockage.

In previous bio-electrospray studies, we and others demonstrated the ability of this platform technology to handle a wide range of living cells in combination with biomolecules.^2,3 Those studies showed not only the ability of this platform to handle such complex and sensitive materials but also to do so without having any negative effects brought on to them from a molecular level upward.^2,5,6 Those studies demonstrated a cell viability of ∼70%, which was comparable to those control cells (cells that were not exposed to bio-electrosprays). Interestingly, these studies were the first to explore flow cytometry for assessing cell viability in the jet-based cell handling/printing community. Note that flow cytometry is a technology used in most pathology laboratories in hospitals globally; thus, these readouts are clinically accepted.⁷ Although the 3D bioprinting community has claimed to have nearly 100% viable cells post-jetting (predominantly assessed via a combination of staining and microscopy), recent studies on cell viability have elucidated the negative effects brought on to the processing and processed biomolecules/cells.⁸ In fact, the biomolecules and cells have been shown to significantly incur molecular/cellular damage/death post-jetting.⁸ 3D printing inflicts damage to both biomolecules and cells, as the technology is hampered by its operation and use of narrow inner bore needles (which are required for controlling residue resolution), which shear cells within them. Larger inner bore needles could be used, but this would significantly affect the residue resolution. Since our early investigations incorporate cytometry, a flow cytometry study has been carried out on 3D bioprinted cells; unfortunately, the cells have not been fully labeled with established molecular probes for identifying important cellular granularity features for understanding cellular kinetics such as apoptosis.⁸ The reader should note that post-3D bioprinted cells have not undergone any karyotypic/genetic studies, as those undergone by bio-electrosprays.⁶ 

Our studies have shown not only the ability of bio-electrosprays to jet cells without damaging them but also living reconstructs generated via bio-electrosprays to have been transplanted into hosts, which have not shown any rejection to demonstrating bio-electrospray's ability to generate both healthy and diseased biological models.⁹ In our previous work with sperm, we reported the ability to bio-electrospray human sperm and demonstrated their viability using computer-assisted sperm analysis (CASA).¹⁰ In those studies, we demonstrated the equipment set-up and how to stabilize the bio-electrospraying process during the jetting of human sperm, which is a challenge as bio-electrosprays operate with electric fields, and biological suspensions contain high concentrations of ions/conducting molecules, which are part of their basic metabolic requirements. Our previous work also demonstrated the ability to bio-electrospray whole fertilized embryos.² In this work, the question of whether bio-electrosprayed sperm are functionally viable is addressed by CASA and flow cytometry, and we further investigated whether the post-jetted sperm retain their capacity to fertilize oocytes.

The bio-electrospraying equipment used in these studies is similar to those we have explored in our previous investigations. In summary, we explored a high voltage DC power supply (FP-30, Glassman Europe Ltd, United Kingdom), which allowed the application of +30 kV to the stainless-steel needle with an inner bore diameter of ∼3 mm and an outer diameter of ∼5 mm. Silicone tubing was used to connect the charged needle to a 10-ml syringe, which was infused using of a motorized pump (PHD 4400, HARVARD Apparatus Ltd, United Kingdom). A ring grounded electrode was centrally placed approximately 10 mm below the exit of the needle, which held a sterile Falcon tube. A wide operational space was investigated for these studies, but we decided on the parameters of 10 kV for an applied voltage and a flow rate in the regime of 10⁻⁹ m³s⁻¹. These jetting conditions saw the generated droplets collected directly into the sterile Falcon tubes.

Bovine sperm stored in liquid nitrogen as straws (straw volume 250  $μ$l containing ∼20 × 10⁶, supplied by UKSIRES, United Kingdom) were thawed in a beaker containing tap water at ∼37 °C for ∼30 s. The thawed straws were subsequently cut at both ends, and the sperm was collected into sterile Falcon tubes. The collected sperm was then subjected to density gradient centrifugation. Briefly, sterile Falcon tubes were filled with PureSperm 80% (v/v) colloidal silica and topped with PureSperm 40% (v/v) colloidal silica. The semen was subsequently added on top, dropwise, and subjected to centrifugation at 300 g for 20 min. Post-centrifugation, the layers, including seminal plasma, and the upper and lower layer rafts, were all removed from the Falcon tube. These layers were removed as they predominantly contain immature, immotile, and dead sperm, debris, epithelial cells, and others. The sperm cell pellet at the base of the tube was then resuspended into four different equal aliquots suspended in two sperm friendly media, namely, BO-SEMENPREP (IVF Limited T/A IVF Bioscience, United Kingdom) and PureSperm Wash (PSW-100, Nidacon International AB, Mölndal, Sweden). Subsequently, they were marked as either controls or those samples for bio-electrospraying (either sample had a sperm count of ∼1 × 10⁶ sperm/ml).

The IVOS II (Hamilton Thorne, MA) CASA system was used for accurate and automated sperm analysis. Semen quality assessment for sperm count and concentration; motile is categories: rapid progression (A), slow progression (B), non-progressive motile (C), and immotile (D) according to the WHO manual for semen analysis.¹¹ In addition, the CASA provides detailed kinematic values for each cell and the kinematic profiles for the entire population, including ALH (amplitude of lateral head displacement), VAP (average path velocity), VCL (curvilinear velocity), and VSL (linear velocity).

This technology has been around since the 50s and is widely used in both clinics and hospitals around the world to identify the dynamic health of cells to a plethora of other cellular components, molecules, and functions. In these investigations, we explored a BD LSR Fortessa X-20 (BD Biosciences, Wokingham, United Kingdom) cytometer equipped with both a Blue (488 nm) and a UV (355 nm) laser. These lasers allowed us to identify the stains we employed to label the different aliquots of sperm, thus allowing to ascertain populations of live and dead spermatozoa present in a given aliquot.

Several bovine spermatozoa straws (CANM11596161 021115 250H001123; commercial grade supplied by UKSIRES, Devon UK), stored in liquid nitrogen, were thawed in a beaker holding water at a temperature of ∼37 °C. After approximately 30 s, the straws were cut at both ends and the sperm was collected into sterile Falcon tubes. The sperm was subsequently passed through a 20-min density gradient centrifugation in PureSperm 40/80 solution (PSK-20, Nidacon International AB, Mölndal, Sweden) at 300 g to separate live and motile sperm. Consequently, the sperm pellet was washed through 10-min centrifugation at 300 g in PureSperm Wash solution (PSW-100, Nidacon) to wash the beads, and the pellet was split into four equal aliquots. Two aliquots accommodated the sperm suspended in BO-SEMENPREP (IVF Limited T/A IVF Bioscience, United Kingdom), and the remaining two were suspended in PureSperm Wash. From each sample, one aliquot was recorded as a control and the other was passed through the bio-electrospray process at an applied voltage of ∼10 kV and flow rate in the 10⁻⁹ m³s⁻¹ regime, over an electric field of ∼1 kV/mm. The post-bio-electrosprayed sperm was directly collected into sterile Falcon tubes and analyzed together with their controls, for their kinetics, motility, and hyperactivation using the IVOS II Hamilton-Thorne Research CASA system (Beverly, MA). Following the assessment of samples using the CASA system, a large majority were reported progressively motile in all the bio-electrosprayed [34.9% category A, 8.6% category B, 4.5 category C, and finally 52.1 in category D of sperm motility, Fig. 1(a)] and control samples (39.9% category A, 8.1% category B, 5.5 category C, to finally 46.4 in the motility of sperm, Fig. 1(a)], with no statistical significance noted between the two groups. Further analysis of all velocity motion parameters of spermatozoa again highlighted no significant differences between bio-electrosprayed and control samples [Fig. 1(b)]: average velocity path (VAP, 51.2% vs 48.3 μm/s, respectively, NS), curvilinear velocity (VCL, 109.5% vs 100.1 μm/s, respectively, NS), straight line velocity (VSL, 36.7% vs 30.9 μm/s, respectively, NS), and amplitude of lateral head displacement (ALH, 6.9 vs 6.6 μm, respectively, NS), and no significant differences in terms of proportion of spermatozoa that are hyperactivated [45.1% and 48.0%, respectively, NS, Fig. 1(c)].

The bovine sperm survival characteristics as assessed with the CASA system after bio-electrospray were similar to our previous work, published on bio-electrosprayed human semen.¹⁰ However, in this work, we further dove into assessing the viability of sperm, through their interrogation via flow cytometry. Similarly, to those sperm aliquots prepared for CASA analysis, four fresh aliquots were prepared from the same bull and batch for flow cytometry analysis. Two aliquots as before were bio-electrosprayed and two remained as controls. Bio-electrospraying was carried out at the same operational conditions as those samples exposed and analyzed by the CASA system. The controls and the bio-electrosprayed samples were stained using 10 μg/ml Hoechst 33342 (ThermoFisher Scientific, Cheshire, United Kingdom) and 10 μg/ml propodeum iodide (ThermoFisher Scientific, Cheshire, United Kingdom). Prior to analyzing cells by way of flow cytometry, many sperm samples, containing either live or dead (exposed to 70% ethanol) sperm cells, were labeled with both staining dyes and passed through the cytometer for identifying live and dead cell populations, respectively, and for finalizing gating regions. Simultaneously, smears of sperm were prepared from control and post-bio-electrosprayed sperm pellets on Superfrost glass slides (Fisher Scientific), and sperm pellet treated with 70% ethanol was used as positive control. Slides were air dried for 1 h before fixation for 20 min in 4% buffered formalin solution. The slides were then stained with a mixture of 10 μg/ml Hoechst 33342 and 10 μg/ml propodeum iodide for 5 min before washing with PBS and mounting with antifading Vectashield Mounting Medium and observing under a fluorescence microscope (Model EVOS M5000, ThermoFisher Scientific, Cheshire, United Kingdom). Damaged or dead sperm will take propidium iodide and appear red [Fig. 2(a)], whereas live sperm will only take Hoechst and appear blue [Fig. 2(b)]. Note in panel (2a) that majority of cells appear dead post exposure to ethanol. Panel (2b) represents sample sperm taken from either a control or post-bio-electrosprayed pellet.

Following these studies, the controls and post-bio-electrosprayed samples were assessed through cytometry. Figures 3(a) and (b) demonstrate representative dot plots of control samples. Panels (c) and (d) of Fig. 3 show the characteristic dot plots of those post-bio-electrosprayed sperm cells. Note in Fig. 3, panels (a) and (c)] are control and bio-electrosprayed sperm samples suspended in BO-SEMENPREP, respectively. Panels (b) and (d) are control and bio-electrosprayed sperm samples suspended in PureSperm Wash, respectively. From Fig. 3, we are unclear as to why the PureSperm Wash resulted in a small decrease in each population when compared to those populations suspended in BO-SEMENPREP. We have currently initiated steps to ascertain the reason why this is the case. More importantly, the dot plots (Fig. 3) through flow cytometry demonstrate the two populations, namely, controls and the post-bio-electrosprayed samples that are comparable in their densities for both suspension media.

Dot plots of Fig. 3 further support the CASA data and show that a large population of sperm cells are motile and viable post-bio-electrospraying in comparison to controls. In addition, sperm fertilizing capacity in controls vs post-bio-electrosprayed was assessed by in vitro fertilization of in vitro matured bovine oocytes. In this regard, ovaries were collected from an abattoir and transported to the lab in warm (37 °C) sterile PBS within 2 h after slaughter. Oocytes were harvested through aspiration of ovarian follicles (3–8 mm in diameter), washed in oocyte washing medium (M199 supplemented with 10% fetal calf serum and 1 mM HEPES buffer), and cultured for 24 h in a humidified 5% CO₂ in air incubator for in vitro maturation before fertilization with the control and post-bio-electrosprayed sperm cells. It should be noted, for fertilization studies carried out in these investigations, we employed only those control and post-bio-electrosprayed sperm cells washed in PureSperm Wash medium and suspended in BO-SEMENPREP, as it contained a larger population of living cells. BO-IVF medium (IVF Bioscience) containing 1 × 10⁶ live sperm was used to fertilize the oocytes after 24 h of co-incubation in a humidified 5% CO₂ in air incubator (Fig. 4). No statically significant differences were observed in the cleavage rate to two-cell plus on day 2 post-fertilization (54.2 ± 3.5% in control and 54.7 ± 4.4% in sprayed group).¹² This indicates that sperm structure (acrosomal membrane) and molecular integrity (both biochemically and physiologically) have remained intact post-BES, as their ability to fertilize the oocytes was not compromised. The acrosomal membrane is present on the sperm head and contains enzymes that facilitate the entry of capacitated spermatozoa into the oocyte during fertilization. Premature acrosomal reaction and release of the enzymes prior to sperm–oocyte interaction result in fertilization failure.¹² Post-incubation of the cleaved embryos from control and post-bio-electrosprayed sperm cells was analyzed with the aid of a time-lapse embryoscope (Vitrolife, Gotenburg). Using the time-lapse embryoscope over a period of ∼8 days, we were able to see all the stages of fertilized oocytes undergoing all expected embryological development according to the International Embryo Transfer Society (IETS) guideline (Fig. 4).¹² Importantly, development to the expanded blastocyst stage (22.9 ± 1.2% in BES embryos vs 20.3 ± 2.3% in control) was not affected. Similarly, no differences were observed in the timeline and pattern of cell division of the cleaved embryos (first cleavage 28.0 ± 0.16 in control vs 29.1 ± 0.7 h, and development to blastocyst stage 174.0 ± 1.2 vs 171.1 ± 0.7 hours post fertilization).

These studies demonstrate for the first time that post-bio-electrosprayed bovine sperm cells are comparable to controls and remain both motile and viable. Furthermore, these investigative studies elucidate that these viable spermatozoa (both controls and post-bio-electrosprayed) have retained their capacity to fertilize oocytes. It is important to note that these cells were able to fertilize oocytes and that these fertilized embryos followed all the expected stages of pre-implantation development. These studies demonstrate this platform technology as a safe jetting technology for the direct handling of living biological materials, such as sperm. Similar results were observed for those spermatozoa exposed to aerodynamically assisted bio-jets. The authors wish to develop these technologies as novel sperm and oocyte handling approaches for controlled distribution of such living materials in 3D proximity, for their use as models, and for studying a wide range of basic biology cues, to its utility for analyzing, sorting, and preserving sperm cells.

SNJ wishes to thankfully acknowledge the Royal Society of the United Kingdom for funding the BioPhyisics Group at UCL.

The authors have no conflicts to disclose.

Ali A. Fouladi-Nashta: Formal analysis (equal); Investigation (equal); Methodology (equal); Writing – review & editing (equal). Fataneh Ghafari: Formal analysis (supporting); Investigation (supporting); Methodology (equal); Writing – review & editing (equal). Walid E. Maalouf: Formal analysis (equal); Investigation (supporting); Methodology (equal); Writing – review & editing (supporting). Natalie J. Werling: Data curation (equal); Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Writing – review & editing (supporting). Suwan N. Jayasinghe: Conceptualization (lead); Data curation (equal); Formal analysis (equal); Investigation (equal); Methodology (equal); Project administration (equal); Validation (equal); Visualization (equal); Writing – original draft (equal); Writing – review & editing (equal).

The data that support the findings of this study are available within the article.