Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Fabrication of the pod and garage
Our device is comprised of 2 components, the Pod and Garage, which were designed using 3D modeling software (SolidWorks®, Dassault Systèmes SE, Paris, France). Fabrication of the devices was performed using two-photon polymerization technology, using a Nanoscribe Photonic Professional GT printer (Nanoscribe GmBH, Eggenstein-Leopoldshafen, Germany).
The 3D design files (standard tessellation language;.STL) were imported into Describe software (Nanoscribe, Karlsruhe, Germany). The fabrication parameters (i.e., laser printing pattern, structure fill: solid or shell and scaffold) were set into a fabrication job file (.GWL). Fabrication was then performed by direct laser writing. The writing speed and laser power were set to 75 MHz and 75%, respectively.
A 25 × microscope objective, numerical aperture 0.8, was used to focus the laser beam into the sample. The Pods and Garages were fabricated onto a substrate (50 × 50 × 0.55 mm indium-tin oxide glass slide, Fluke Australia Pty Ltd., Baulkham Hills, NSW, Australia). Prior to printing, the glass substrate was rinsed with ethanol then isopropyl alcohol and dried with compressed air.
An IP-S photoresist resin (Nanoscribe GmBH) was used to fabricate the Pods and Garages. Following printing, the excess unfabricated resin was removed by washing in isopropyl alcohol for 8 min, followed by 8-min development (SU-8 developer; Nanoscribe GmBH), and then dried using compressed air. Next, the substrate was submerged in 5% 7X-O-Matic cleaning solution (MP Biomedicals, Solon, OH, USA) overnight at room temperature (RT). Following fabrication, the Pods and Garages were carefully removed from the glass substrate. Once removed, the Pods and Garages were washed three times in 5% 7X-O-Matic cleaning solution at RT. This was followed by three overnight washes in filtered phosphate-buffered saline (PBS; one tablet per 200 mL of Milli-Q water) at RT. The Pods and Garages were stored in PBS at 4 °C until use.
All experiments were approved by the University of Adelaide Animal Ethics Committee (M-2019–008) and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.
Female (pre-pubertal, 3–4 weeks old) and male (6–8 weeks old) CBA × C57BL/6 first filial generation (F1) mice (CBAF1) were obtained from the University of Adelaide Laboratory Animal Services and maintained under 12-h light to 12-h dark cycle with rodent chow and water provided ad libitum.
Media for gamete and embryo handling and culture
All gamete and embryo culture took place in media overlaid with paraffin oil (Merck Group, Darmstadt, Germany) at 37 °C in a humidified incubator with 5% O2 and 6% CO2 balanced with N2. For the Pod and Garage treatment groups, a single Garage and 3 individual Pods were placed into a drop of culture medium using fine forceps. Culture dishes for all treatment groups were then pre-equilibrated for at least 4 h prior to use. Oocyte and embryo handling were carried out on a heated stage with the temperature set at 37 °C. Mouse tissues were collected in Research Wash Medium (IVF VET Solutions, SA, Australia) supplemented with 4 mg/mL low fatty acid bovine serum albumin (BSA; MP Biomedicals, Albumin NZ™, Auckland, New Zealand). Research Cleave Medium (IVF VET Solutions) was also supplemented with 4 mg/mL BSA and used for embryo culture (manufacturer’s recommended density = 2 μL/embryo). Embryos were cultured from the zygote to blastocyst stage in a single-step culture system (i.e., no media change occurred on day 3 of development).
Isolation of mouse cumulus oocyte complexes
Pre-pubertal female mice were injected intraperitoneally (i.p.) with 5 IU equine chorionic gonadotrophin (eCG; Folligon; Pacific Vet Pty Ltd., Braeside, VIC, Australia) followed by 5 IU (i.p.) human chorionic gonadotrophin (hCG; Pregnyl; Merck, Kilsyth, VIC, Australia) 46–48 h later. Mice were culled via cervical dislocation, and the ampullae of the oviducts dissected in warmed Research Wash Medium. Ovulated cumulus oocyte complexes (COCs) (14–16 h post-hCG) were isolated by puncturing the ampullae in warmed Research Wash Medium. The isolated COCs were briefly incubated in hyaluronidase (6.1 μM) diluted in warmed Research Wash Medium for 1 min to remove cumulus cells with the aid of gentle pipetting.
Isolation of mouse presumptive zygotes
Pre-pubertal female mice were injected with eCG (5 IU; i.p.) followed 46–48 h later by 5 IU if hCG (i.p.). Females were then paired overnight with males, with mating confirmed the following morning by the presence of a copulation plug. Female mice were culled via cervical dislocation and the ampullae dissected to isolate presumptive zygotes (PZs) (22–24 h post-hCG). Cumulus-enclosed PZs were incubated in hyaluronidase (6.1 μM) diluted in warmed Research Wash Medium for 1 min to remove cumulus cells with the aid of gentle pipetting.
Analysis of pod and garage embryo toxicity
The toxicity of the Pod and Garage was assessed using a standard mouse embryo assay (MEA) that used both negative and positive controls, with a certificate of assessment provided (IVF VET Solutions, SA, Australia) . The Pods and Garages were soaked in protein-free MEA medium and incubated overnight at 37 °C in a humidified incubator with 5% O2 and 6% CO2 balanced with N2. Embryo culture drops (10 PZs/20 μL) were then prepared using the MEA medium utilized to wash the Pods and Garages. In addition to the MEA test, embryo culture from the PZ to the blastocyst stage was conducted within Pods docked in a Garage (three PZs docked individually in 3 Pods and a Garage/10 μL) using Research Cleave Medium supplemented with BSA. Fertilization rate was scored 24 h later, with embryos then allowed to develop to the blastocyst stage within Pods and a Garage. At 96 h post fertilization, embryos were considered on-time if at the blastocyst stage (i.e., having a blastocoel cavity ≥ two-thirds the size of the embryo; or expanded; or hatching).
Analysis of DNA damage/repair in cultured blastocysts (phosphorylated-histone-H2A.X; γH2A.X)
Unless otherwise stated, all immunohistochemistry procedure was carried out at RT. Following either standard embryo culture or embryo culture within the Pods and a Garage, embryos were stained with γH2A.X to assess for double-stranded DNA repair . The blastocysts were fixed in 200 μL 4% paraformaldehyde diluted in PBS (w/v) for 30 min following fixation, blastocysts were washed with PBV (0.3 mg/mL polyvinyl alcohol diluted in PBS) and permeabilized for 30 min in 0.25% (v/v) Triton X-100 in PBS. Blastocysts were then blocked for 1 h with 10% goat serum (v/v; Jackson Immuno, PA, USA) diluted in PBV. Following blocking, blastocysts were incubated overnight in the dark with anti-γH2A.X primary antibody (Cell Signaling Technology, MA, USA) at a 1:200 dilution with 10% goat serum in PBV (v/v). A negative control was included where embryos were incubated in the absence of the primary antibody. Next, embryos were washed three times in PBV before incubation for 2 h in the dark with anti-rabbit Alexa Fluor 594-conjugated secondary antibody (Life Technologies, Carlsbad, CA) at 1:500 dilution with 10% goat serum in PBV (v/v). Embryos were then counterstained with 3 mM of 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, MA, USA). Finally, embryos were washed three times in PBV and transferred onto a glass microscope slide with DAKO mounting medium (Dako Inc., CA, USA) and enclosed with a coverslip using a spacer (Thermo Fisher Scientific, MA, USA). Embryos were imaged using an Olympus Fluoview 3000 confocal microscope (Olympus Life Science, Tokyo, Japan). Images were captured at 60 × magnification, using the imaging channels Alexa Fluor 594 (red) for γH2A.X (591/614 nm) and DAPI (blue) for DNA (358/461 nm). A z-stack projection for each blastocyst was generated using images captured at 4-μm intervals. The same imaging parameters were kept for each replicate. The intensity of γH2A.X immunostaining was quantified using Fiji ImageJ software (National Institute of Health, MD, USA).
Comparing standard microinjection vs microinjection within the Pods and a Garage
Microinjection process was assessed to compare technical components for standard microinjection vs within the Pods and a Garage under a micromanipulator. Microinjection was performed in a 60-mm petri dish lid (Falcon, Corning, In Vitro Technologies, VIC, Australia). The microinjection drops were prepared with warmed Research Wash Medium (5 × 10 μL drops at the center of the dish overlaid with paraffin oil). The microinjection dish was pre-equilibrated on a heated stage at 37 °C for at least 4 h before use.
Mouse oocytes were loaded into the microinjection drops to compare standard microinjection (three oocytes/drop) and microinjection within the Pods and a Garage (three oocytes within Pods and a Garage/drop). For standard microinjection, the holding pipette (inner diameter: 17 μm; outer diameter: 80 μm; bevel: 30°; Cook Medical, PA, USA) and injection pipette (inner diameter: 5 μm; outer diameter: 7 μm; bevel: 20°; Cook Medical) were mounted into the micromanipulators. Conversely, for microinjection within the Pods and a Garage, only the injection pipette was used. For standard microinjection and microinjection within a Pod and Garage, the orientation of the oocyte with respect to the polar body was adjusted to either the 6 or 12 o’clock position by placing the injection pipette above the oocyte (in proximity to either the top or the bottom of the oocyte) and moving the pipette along the z-axis.
Microinjection of PZs with fluorescent microspheres
To demonstrate the application of the device for ICSI, microinjection of 4-μm fluorescent microspheres (Invitrogen, Thermo Fisher Scientific, MA, USA) was performed in PZs within the Pods and a Garage. Microinjection of PZs with fluorescent microspheres occurred under a Nikon Eclipse TE2000-E inverse microscope (Nikon Instruments Inc.) equipped with a Tokai Hit ThermoPlate set at 37.5 °C.
Only the injection pipette was loaded into the micromanipulator and used to perform microinjection. The microspheres were aspirated from a separate drop consisting of warmed Research Wash Medium and were then individually injected into each PZ.
Following microsphere microinjection, PZs were transferred from within the Pods and a Garage into pre-equilibrated 2 μL Research Wash Medium on a glass-bottomed confocal dish (Cell E&G, Houston, TX, USA) overlaid with paraffin oil and imaged under the Olympus Fluoview 10i confocal microscope. The fluorescent microspheres were then visualized using the red fluorescence channel (660/680 nm). The microinjected PZs were then cultured in Research Cleave Medium and were allowed to develop to the blastocyst stage.
Assessment of multiple oocyte microinjection capability within Pods and Garage
The feasibility of microinjecting multiple oocytes within the Pods and a Garage was then tested and compared to microinjecting multiple oocytes using the standard procedure. Microinjection was performed on a microinjection dish with three oocytes loaded per drop for standard microinjection and for microinjection in our device (three oocytes/3 Pods docked in a Garage) within a separate drop. The microinjection drop size for standard microinjection and microinjection within the Pods and a Garage was 10 μL. Under standard microinjection, oocytes were placed into the drop using a pulled glass pipette. For microinjection within the Pods and a Garage, oocytes were loaded into a Pod (1 oocyte per Pod) using a pulled glass pipette. Each Pod was then docked within a Garage using fine forceps.
During standard microinjection, the holding and injection pipettes were utilized to locate and hold individual oocytes prior to microinjection manually. Subsequent microinjection of oocytes was performed after the injected oocyte was released and separated from the non-injected oocyte cohort within the same drop. This process was also performed manually using the holding and injection pipettes. Conversely, for microinjection within the Pods and a Garage, only the injection pipette was utilized. Oocytes were arranged next to each other within our system prior to microinjection. The micromanipulator stage was controlled manually to lead the injection pipette into the non-injected oocyte through the injection pipette channel of the Pod. The micromanipulator stage was then used to facilitate microinjection. Subsequent microinjection of non-injected oocytes was also performed within adjacent oocytes within the remaining Pods docked in a Garage using the micromanipulator stage.
Comparative analysis of time taken to microinject oocytes within the Pods and Garage vs standard microinjection
Three mouse oocytes were preloaded into individual Pods that were then docked within a Garage. Quantification of time required for individual parameters of the microinjection procedure and comparison between standard and microinjection within the Pods and a Garage was performed. Key parameters considered for this experiment were setting up of pipettes (holding and injection pipettes), holding oocytes before microinjection, and injecting each oocyte. Each parameter within the entire procedure was measured individually (Fig. 5b).
All statistical analyses were performed using GraphPad Prism 8.0 for Windows (GraphPad Software, San Diego, CA). Embryo development data were arcsine transformed prior to statistical analysis. All experimental data were tested for normality to determine whether a parametric or non-parametric test should be used. Statistical analyses were performed using a Student’s t-test as described in the figure legends. A P-value < 0.05 was considered statistically significant.