Key words

1 Introduction

The lumen of the oviduct is a complex environment which supports fertilization of oocytes, early embryo development, and facilitates embryo transport to the uterus. This transport occurs due to the flux generated by cilia and peristaltic movements from the contraction of the smooth muscle layer. The combination of muscular and cilia actions over tubal crypts generates a turbulent flow toward the uterine horn (1). This turbulent progressive movement is responsible for homogenization of tubal secretion, dilution of undesirable metabolites, and facilitation of gamete interaction (1). The velocity of tubal flow and the frequency of ciliary movement is estimated to range from 6.5 to 29 μm/s and 5 to 20 Hz, respectively (2). This dynamic microenvironment provides mechanostimulation of the embryo (3). Despite the lack of understanding on how embryo mechano-sensitivity is mediated, it is possible, that as in other cells, mechanostimulation modulates gene expression and cell fate (4, 5).

Even though in vivo embryo development occurs in a dynamic environment, embryo culture in static conditions is possible. Indeed, static systems for in vitro embryo culture are the most common methods for the growth of mammalian embryos and they are widely adopted in human infertility treatments and the production of livestock. However, studies with animal models show that, in general, in vitro-grown embryos have a lower quality and produce lower pregnancy rates than in vivo-produced embryos. It is known that exposure to the tubal/uterine environment during early development modulates metabolic pathways of embryos (6–9), which suggests that perhaps current static conditions used in vitro may not be providing an ideal culture environment.

Due to the evident discrepancy between the environment of static culture systems and of the tubal/uterine environment, researchers have theorized that embryo culture systems utilizing fluidic dynamics could recapitulate beneficial conditions and stimuli received by the embryo in in vivo conditions. This chapter reviews several methods to obtain fluidic dynamic culture and the materials and methods necessary to produce dynamic funnel devices in order to culture embryos using this innovative approach.

1.1 Generation of a Dynamic Fluid Environment

Several articles have discussed the potential applications and advantages of dynamic environments for embryo culture obtained with microfluidics (10–14). Although these reports were optimistic and drew a promising perspective, a limited number of publications actually investigated the effects of dynamic environment on embryo development (15–19). The small number of investigative reports is likely due to the relative complexity in building systems able to combine embryo safety, ease use, and well-established physical and chemical requirements of embryos with the generation of appropriate fluid dynamics.

In general, reported methods to generate fluid dynamics can be divided into three categories: (1.2) platform/dish movement, (1.3) wave fluid movement, and (1.4) designed fluid dynamics. The adoption of any one of these methods should take into account the primary goal pursued: embryo culture with an added benefit of improved development, analysis, and/or production of healthier offspring. For example, generation of fluid dynamics with platform movement or random fluid movement might be suitable for the production of embryos (16, 17), but may not allow embryo assessment beyond traditional morphological evaluation. The ­election of a ­programmable designed fluid dynamic system would allow embryo culture with fluid movement, morphological assessment of the embryos, but it also potentiates integration of other technologies to the system, such as real-time analysis of embryo metabolome and secretome (20). In this manner, the adoption of one of these methods for the production of fluidic dynamics should be based on the needs and goals of each embryology laboratory.

1.2 Platform/Dish Movement

Media in traditional embryo culture are usually held in plastic ­culture dishes covered with oil to avoid evaporation. This dynamic culture strategy uses the same plate system, but varies from traditional static culture in that, while in the incubator, rotation or ­tilting of the platform in which culture dishes are kept creates fluidic movement (17). This alters the environment in which the embryo is cultured. The utilization of dish movement to create a dynamic environment for embryo culture was reported over 35 years ago (21), although the authors used a short period culture and this early study failed to consider shear stress exerted on embryos. Embryo tolerance and resilience to shear stress were determined more recently (22).

Platform movement to generate dynamic fluid in human embryo culture was examined recently by Matsuura et al. (17). The fluid dynamic was attained by tilting the platform to produce embryo and fluid movement with a radial velocity of 1° per second up to 10°. This setting produced an embryo rolling speed of 0.3 mm/min and a shear stress of 1.5  ×  10−4 dyn/cm2. Although there was no significant increase in the rate of blastocyst formation, the number of blastomeres in embryos cultured in the tilting embryo culture system was higher than in the static culture. Whether this increased numbers of blastomeres in human embryos subjected to tilting embryo culture will convert to increased pregnancy rates still needs to be determined in clinical trials. Similar effects of tilting embryo culture system on the number of blastomeres per blastocyst were observed in porcine and murine embryos (16, 17).

Tilting platforms also were used for the movement of embryos in microchannel devices (18). Devices containing microchannels with a series of constrictions were tilted to move bovine embryos through areas of varying width. It was proposed that this resulted in simulating peristaltic forces on embryos and mimic tubal peristalsis. This system remains to be optimized and tested in order to determine the range of beneficial forces applied to the embryos (18).

1.3 Wave Fluid Movement

Mechanical stimulus through vibration seems to increase the proliferation rate and behavior of some types of somatic cells (23–25). Platform vibration can be used to modulate embryo development in vitro through fluid and embryo movement (26, 27). Pig oocytes maturated in vitro under vibration (20 Hz) for 5 s every 60 min produced higher blastocyst rates than those in static culture; however, beneficial effects of embryo vibration were not observed when embryos originated from oocytes matured under static conditions (26). This might indicate that, at least for this species, vibratory movement might have a positive impact during oocyte maturation (26). More recently, wave fluid movement was applied to human embryos in a similar fashion, but with a higher frequency (44 Hz) (27). The authors state that in their experience the application of 44 Hz vibration for 10 s every 60 min resulted in increased rates of blastocyst formation and pregnancy (27). The underlying mechanisms of the effects imposed by vibratory stimulation are still unknown.

1.4 Designed Fluid Dynamic

Designed fluid dynamics are obtained by forcing fluid through a microscopic and geometrically restrained area, commonly through channels. Microfluidics devices described for embryo culture include the use of (Subheading 1.4.1) gravity gradients, (14, 28), syringe pumping (29), or (Subheading 1.4.2) peristaltic movement produced by Braille pins over a flexible surface (15).

1.4.1 Gravity Gradient and Syringe Pumping

Gravity gradient and syringe pumping of media through a microchannel are simple methods to generate microfluidic dynamics. Generally, devices designed for embryo culture in fluid dynamic systems have a single channel with barriers (30, 31) or simple arrays that can be produced through photolithography or micro-molding of thick or hard elements because these methods do not require flexible membranes (28). Usually, they have relatively simple construction and operation, and can accommodate more than one function. Indeed, microchannel devices have been used for cumulus cell and zona pellucida removal and embryo culture (14).

The great advantage of these devices are their simplicity, although there are some important considerations regarding the generation of shear stress, wash out of desirable autocrine factors and also the risk of embryo entrapment into the system. Embryos confined in a microchannel might be submitted to a unidirectional flow of up to 100 nL/s at an average velocity of 2 mm/s (28) and forces up to 10−7 N. Further modeling and simulations with this type of channel indicate that embryos in these devices are submitted to shear stress and washout of growth factors and cytokines secreted by the embryos (15).

Detrimental and time-dependent effects of shear stress on embryos were elegantly demonstrated by Xie et al. (22). Embryos submitted to shear stress of 1.2 dyn/cm2 had increased activity of MAPK 8/9 triggering apoptosis. These effects were noticeable after 6 h of submission to the shear force. At this stage, several embryos were able to recover from stress; however, the proportion of embryos failing to recover after the exposure to shear forces increased in a time-dependent manner and after 12 h none of the embryos were able to recover from the stress (22).

Embryos grown in microchannel devices with fluid flow are subjected to a lesser degree of shear forces than those reported in Xie et al. (22). However, it is important to recognize there period of exposure are significantly longer and therefore may impact overall shear stress and account for compromised development. (19, 28). In some instances, these reports indicate an increase of blastocyst production in microchannels culture systems (19, 32), yet data are needed in relation to pregnancy rates, number of blastomeres per embryo and subcellular changes like increased activity in MAPK 8/9.

1.4.2 Peristaltic Movement

Programming actuation of sequential Braille pins that can press against and close a flexible membrane covering a microfluidic channel can deliver different types of peristaltic flow (33). This type of flow generation permits the control of frequency of fluid displacement and speed of fluid. Many times the material utilized for ­construction of these microfluidic devices is the polymer polydimethylsiloxane (PDMS), because it is easy to prototype, is gas permeable, flexible, and its optical properties allow microscopic observation (15, 18, 34, 35). However, the requirement for a flexible membrane needed for Braille pin actuation can lead to considerable evaporation of media and shifts in media osmolality (36).

To circumvent evaporation and increase in osmolality, several thicknesses (0.1–10 mm) of flexible PDMS membranes and different impermeable treatments were investigated. Thicker PDMS membranes had less evaporation while PDMS membranes of 0.1 mm and 0.2 mm yielded the maximum evaporation and large osmolality increases that were detrimental to embryo development. Osmolality changes of more than 300 mosm/kg, observed in microfluidic devices with microtunnel bottoms of 0.1 mm of PDMS, impaired the development of embryos to the blastocyst stage in comparison to thicker PDMS walls (PDMS 1 mm  =  72 % and PDMS 10 mm  =  67 %) and control devices with glass bottoms (67 %) (36). This presented a dilemma of needing a thin layer for Braille pin actuation, yet needing to prevent the culture environment from being detrimentally affected by evaporation and osmolality shifts not supportive of embryo development. Thus, it became necessary to treat PMDS to yield a flexible yet impermeable membrane. It was determined that coating the thin walls of PMDS in fluidic devices with 2.5 μm film of parylene prevented osmolality shift and increased the rate of blastocyst formation in comparison with uncoated devices (36).

The microfunnel shape to hold embryos within a microfluidic device with peristaltic flow was tested with extensive modeling and simulation. Embryo culture in microfunnels diminishes shear stress compared to embryos within microchannels with dynamic flow. Additionally, the microfunnel increases the concentration of autocrine factors around embryos and provides mechanostimulation (15). The advantages gained by the dynamic microfunnel can be observed in mouse embryos cultured under pulsatile movement with frequency of 0.1 Hz in prototypes of microfunnel devices. The percentage of hatched blastocysts and the number of blastomeres per embryo were greater after embryo culture in microfunnel dynamic culture system in comparison to microfunnel and petri dish static culture. Furthermore, these findings were followed by implantation and pregnancy studies that found enhanced implantation and ongoing pregnancy rates in embryos culture in the microfunnel dynamic system in comparison to static culture and they closely mirrored rates obtained with in vivo produced embryos (15).

Prototypes of dynamic microfunnel culture devices are undergoing evaluation in the United States and Brazil (37) and initial reports indicate that embryos grown in dynamic microfunnel devices until day 3 of development had lower fragmentation rates in comparison to those kept under traditional static conditions. Furthermore, human embryos grown in dynamic microfunnels were more likely to develop into embryos of good quality (greater than six blastomeres with less than 20 % of fragmentation on day 3) than those cultured under traditional static conditions (37). The effect of dynamic microfunnel devices on human embryo development to the stage of blastocyst and the impact of this technology on implantation and pregnancy rates should be addressed in forthcoming research.

1.5 Final Considerations

Devices for fluid dynamic embryo culture systems can be designed for different functions in an embryology laboratory and several reports demonstrate that different types of devices produce dynamic fluid environments and enhance embryo production. Clearly, these experiments targeted the short-term goal of providing more ­efficient culture systems for clinical IVF laboratories and have demonstrated that advantages can be obtained through numerous technical avenues. Of great potential is the integration of technologies into a single device within the clinical-assisted reproductive technologies laboratory. For this utility, a designed and programmable dynamic culture system will be needed to facilitate numerous aspects of existing gamete/embryo clinical laboratory manipulation and cultivation, as well as allowing new abilities such as real-time bioanalysis, that will collectively improve outcomes in clinical treatment of infertility. This provides the platform for ­laboratory-on-a-chip devices that couple embryo culture to ­real-time analysis of metabolites and/or secreted factors. This type of device might have tremendous applications in the clinical embryology setting to assist embryo selection with criteria based on physiological performance, rather than morphology criteria alone. In fact, the same features that make microfluidic devices attractive to clinical embryology laboratories might make these devices interesting to experimental embryology laboratories. The ability to produce sequential and/or gradient delivery of different chemicals can be used to generate distinctive experimental designs and may lead to further improvements in fields such culture media development or cryobiology.

2 Materials

The following materials and methods relate to the utilization of a prototype system of a dynamic microfunnel device for embryo culture. Adjustments and changes should be incorporated into this protocol for the proper function of this prototype in other laboratories, as well as for the utilization of other in-house built systems for embryo culture based on the technology of microfluidic dynamics (15, 36, 38).

2.1 Materials for Construction of Dynamic Microfunnel Devices

  1. 1.

    PDMS prepolymer (Sylgards 184, Dow Corning).

  2. 2.

    Parylene C (see Note 1).

  3. 3.

    200 μm Glass slide.

  4. 4.

    SU-8.

  5. 5.

    Tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorsilane.

  6. 6.

    Commercial Braille display.

2.2 Materials for Embryo Culture in Dynamic Microfunnel Devices

  1. 1.

    Laminar flow hood.

  2. 2.

    Incubator for embryos.

  3. 3.

    Braille pin platform (see Note 2).

  4. 4.

    Dynamic microfunnel device.

  5. 5.

    Embryo culture media.

  6. 6.

    Pipette for volumes up to 1 mL.

  7. 7.

    Pipette tips with barrier.

  8. 8.

    Small-bore pipette.

  9. 9.

    Pipetting device for small-bore pipettes.

  10. 10.

    Inverted microscope.

  11. 11.

    Stereomicroscope.

3 Methods

3.1 Construction of Dynamic Microfunnel Device

  1. 1.

    Model the design of your device (see Note 3).

  2. 2.

    Compose desired channel structures composed of SU-8 and fabricate them on a thin glass wafer using backside diffused-light photolithography.

  3. 3.

    Cast an 8 mm PDMS prepolymer (1:10 base-to-agent ratio) slab with microchannels and funnels against the SU-8 model.

  4. 4.

    Remove bubbles under vacuum for 1 h (200 mmHg) (see Note 4).

  5. 5.

    Cure the casted 8 mm PDMS prepolymer slabs at 60 °C for 120 min and 120 °C for 30 min.

  6. 6.

    Cast 0.1 mm PDMS membranes as described above.

  7. 7.

    Deposit parylene C (∼2.5 μm) on the back side of 0.1 mm PDMS membranes using a PDS 2010 Labcoater after covering the well side with PDMS.

  8. 8.

    Attach PDMS slab to a thin PDMS-parylene-PDMS membrane.

3.2 Set Up of Dynamic Microfunnel Device and Embryo Culture

  1. 1.

    Dynamic microfunnel devices for embryo culture are gas ­permeable plates and they should be placed into incubators at 37 °C for at least 4 h before loading with culture media (see Note 5).

  2. 2.

    The dynamic microfunnel device can have two microfunnels connected by microchannels. Place media into microfunnels. The media will enter the microchannels and proceed through the channel and into the other microfunnel.

  3. 3.

    Search under inverted microscope or stereomicroscope for bubbles of air in the two microchannels connecting reservoir and culture microfunnels (see Note 6). Microchannels should be clear of air bubbles.

  4. 4.

    If the microchannels contain air bubbles, use a plastic rod to gently press the microfluidic device from the bottom and move the rod in the direction of the microfunnel to drive air bubbles out of the microchannels.

  5. 5.

    Once the dynamic microfunnel device is clear of air bubbles, it should be clipped on top of the Braille pin platform stored inside the incubator (see Note 7).

  6. 6.

    The Braille pin platform should be turned on to initiate flow of media and the whole system should run overnight before placing embryos in culture.

  7. 7.

    Check the dynamic microfunnel cartridge again for the presence of air bubbles before placing zygotes into the culture microfunnel (see Note 8).

  8. 8.

    Remove zygotes from the static culture system with the ­assistance of a stereomicroscope and a small-bore pipette. Place up to five embryos on a culture microfunnel. Replace the dynamic microfunnel cartridge onto the Braille pin platform and lock the safety brackets before placing back into incubator and activating the movement of Braille pins.

  9. 9.

    Embryo should be evaluated on day 3 or 5/6 of culture.

4 Notes

  1. 1.

    Parylene coating can be provided by third party companies specialized in chemical coating as Specialty Coating System.

  2. 2.

    Use commercially available Braille pin platform for e-mail reading (Braillex Tiny from F.H. Papenmeier GmbH & Co. KG, Germany).

  3. 3.

    Theoretical modeling of microfunnel devices should be performed before casting prototypes to ensure that the intended shear stress and chemical diffusions are obtained. The device utilized in Heo et al. 2010 (15) had the following dimensions:

    1. (a)

      Microchannels: length—1.5 mm, width—0.1 mm and height—0.11 mm.

    2. (b)

      Funnel: bottom radius—0.25 mm, top radius—1.77 mm, height—2.63 mm and angle in relation to the flat bottom of the funnel—60 °.

  4. 4.

    Avoid excessive exposure to vacuum to prevent premature curing.

  5. 5.

    Placing the microfluidic device for at least 4 h into an incubator at 37 °C may allow expansion prior to loading with media and is used as a strategy to avoid the formation of air bubbles in microchannels.

  6. 6.

    This procedure has to be carefully performed under a stereomicroscope. The presence of air bubbles should be avoided because they block the movement of media in microchannels and negate the potential benefit of dynamic culture.

  7. 7.

    Incubator should be set up according to standard practices of each clinical embryology laboratory, regulating temperature, and gas environment.

  8. 8.

    Oocyte insemination and determination of fertilization should follow the standard procedures and guidelines of each clinical embryology laboratory.