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From: Methods in Molecular Biology, vol. 254: Germ Cell Protocols, Volume 2:Molecular Embryo Analysis, Live Imaging, Transgenesis, and Cloning

Edited by: H. Schatten © Humana Press Inc., Totowa, NJ

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Mammalian Embryo Culture in a Microfluidic Device

Eric M. Walters, Sherrie G. Clark, David J. Beebe,and Matthew B. Wheeler

1. IntroductionOver the last decade, the use of in vitro production of mammalian embryos

and the utilization of assisted reproductive technologies (ART), such as non-surgical embryo transfer, cryopreservation, and intracytoplasmic sperm injec-tion (ICSI), has increased. However, the efficiencies of ART remain low.Currently, millions of couples in the United States are affected by infertilityand seek treatment. More than 16,000 babies in the United States, each year areborn using ART such as in vitro fertilization (IVF), assisted hatching, and ICSI(1). A critical area for ART is culturing embryos and evaluating their morphol-ogy. There have been some advances made in embryo culture medium;however, the efficiency of embryo culture remains low. Additionally, embryo-handling techniques and tools for culture and evaluation have not changed forseveral years. The growth of miniaturization technologies toward miniaturemechanical and fluidic systems has created opportunities for fresh examina-tion of basic embryo physiology. There are many existing limitations in mam-malian embryo-handling procedures that may be addressed with these newminiaturization technologies.

A new miniaturized technology called the Micro Embryo Culture Chip(MECC), which has been shown to improve in vitro production of mammalianembryos, was recently developed (2–7). The MECC represents a significantparadigm shift from the traditional microdrop culture systems. There are sev-eral advantages to using the MECC for embryo culture. First, the MECC pro-vides a microenvironment that is more in vivo-like. If the embryo-to-medium

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volume ratio is compared in the traditional microdrop system, the ratio is largerwhen contrasted to the estimated ratio in the oviduct. In the MECC, the embryo-to-volume ratio more closely mimics the oviductal environment. Also, theMECC reduces stress to the embryo by reducing handling. During the tradi-tional in vitro production, mammalian embryos can be exposed to more than20 different washes and culture drops. Each wash can impose stress on theembryos by temperature changes, pH, osmolarity, and chemical composition,as well as potentially exposing the embryos to human error. In the MECC, theembryo remains stationary, whereas the medium or chemical composition canbe gradually changed—a situation that more closely mimics the conditions theembryo experiences in vivo.

The MECC (Fig. 1) is designed with a single channel (250 × 1000 μm) witha constriction region in the mid-point of the channel. The constriction regionallows the embryos to “park” in this region while retaining the ability to flowmedium past the embryos to a collection or outlet well. The MECC contains afunnel feature that facilitates loading and unloading of the embryos.

In summary, the MECC is a new tool that may lead to improvements in theunderstanding of basic embryo physiology as well as improved efficiencies of ART.

1.1. Structures of MECC

1. Inlet well: Serves as a reservoir for medium and is the port through which theembryos and medium are loaded into the device.

2. Inlet funnel: Guides embryos directly into the microchannel for easy loading andunloading.

3. Inlet channel: Embryos travel down the inlet channel until they come to rest atthe constriction region.

4. Constriction region: a narrowing of the channel that holds embryos/oocytes inplace while still allowing media to flow though to the outlet.

5. Outlet channel: only medium is present in this part of the device, which leads tothe outlet reservoir.

6. Outlet luer: The Luer serves a dual function, providing a reservoir for down-stream medium, and also to being an airtight Luer connector for a syringe whensuction/pressure is needed in the channel.

7. PDMS: Polydimethylsiloxane (PDMS): is material from which the top of thedevice is made.

2. MaterialsAll chemicals are purchased from Sigma Chemical Company (St. Louis,

MO) unless otherwise stated.

1. MECC (Vitae LLC, Madison, WI). Currently, the devices are not in commercialproduction. Please contact Vitae LLC (608-222-1908 or http://www.vitaellc.com)or Penetrating Innovations (608-845-3270 or http://wwww.pi.ivf.com) for avail-ability of this product.

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Fig. 1. The physical characteristics of the MECC. (A) A photograph of MECCfilled with medium. (B). A schematic diagram illustrating the various structures of theMECC. (Illustration courtesy of Kathryn Haubert, Vitae, LLC, Madison, WI.)

2. Incubator capable of producing 5% carbon dioxide (CO2) in atmosphere (FisherScientific Company, Pittsburgh, PA).

3. Embryo-handling pipet4. 100-mm Falcon 3003 dishes (Fisher Scientific Company).5. 1 × 3-inch Microscope slides (Fisher Scientific Company, Pittsburgh, PA).6. 1-cc syringe (Fisher Scientific Company).7. M2 medium supplemented with 0.4% BSA (fraction V) and M16 medium supple-

mented with 0.8% bovine serum albumin (BSA) (fraction V) used for mouseembryo culture (7).

8. Synthetic oviductal fluid (SOF [8]), Specialty Media, Phillipsburg, NJ) used forbovine embryo culture.

9. North Carolina State University-23 medium (9) used for porcine embryo culture.This can be purchased from Cook Veterinary Supplies, Queensland, Australia.

10. Dual Peel sterilizing film (Fisher Scientific Company).

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11. Mineral oil (embryo tested)12. P1000 pipetman and sterile P1000 tips (Rainin, Woburn, MA).13. Stereomicroscope (Nikon Corporation, Tokyo, Japan).14. 15-mL conical tube (Fisher Scientific Company).

3. Methods3.1. Inspection and Sterilization

1. Inspect the MECC microchannel to make sure there are no obvious obstructions,debris, or other problems.

2. Place individual microchannel into Dual Peel, heat-sealed at both ends (FisherScientific), and autoclave at 121°C for 30 min, and then dry for 20 min.

3.2. Preparation of the MECC

1. Transfer-sterilize the MECC into the lid of a Falcon 3003 Tissue Culture Dish(Fisher Scientific), covering it with the bottom of the dish.

2. Place the MECC into a CO2 incubator for a minimum of 1.5–2 h before fillingwith medium. In addition, the medium to be used must be equilibrated to the gas(5% CO2 in air typically) and temperature (37°C typically but dependent on spe-cies) by incubation in a loosely capped 15-mL conical tube for a similar amountof time before it is loaded into the channels. This preheating and gassing of thechannel and medium dramatically reduces bubble formation when the medium isloaded (see Note 1).

3. After the equilibration period, pipet approx 500 μL of warm medium into theloading well of the MECC. Attach a 1-mL syringe to the outlet Luer, and slowlydraw the medium through the channel until the height of the medium in the inletand outlet ends is approximately equal. If large bubbles (more than one-fourththe width of the channel) are visible, try to displace them by pushing and pullingthe medium back and forth in the channel. Tiny bubbles will likely be absorbed,so temporarily ignore this. Disconnect the syringe, and put the filled channelback into the incubator. Let it equilibrate for a minimum of 1.5–2 h before load-ing embryos (see Note 1)

4. After the medium has equilibrated in the channel, it is very important to check forbubbles before loading embryos.

3.3. Handling of Bubbles in the MECC

1. Determine the size and location of the bubble(s):a. If the bubble is small (Fig. 2), determine if it will interfere with movement of

the cells placed into the channels (see Note 2).b. If the bubble is large (Fig. 2), determine the mobility of the bubble (i.e., can it

be pushed or pulled out of its current position?)

3.3.1. Bubble(S) Occluding the Funnel Entry to the Channeland Bubble-Embryo Interactions

1. Use a long micropipet or pulled pipet tips and remove the bubble.

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Fig. 2. Embryos near the constriction region (CR) with a bubble anterior and poste-rior to the CR.

2. Longer pipettes allow pressure to be applied, which should move or pull thebubble out of the funnel opening without contamination of the equipment owingto excess medium being pulled into the micropipet.

3. Pulled pipets with smaller diameter allow the pipet tip to be placed along side ofthe bubble in the bottom of the funnel and the bubble “plucked” from its occlud-ing position. The bubble will then be allowed to float to the surface of the me-dium in the funnel and removed by the micropipet.

4. Use a push–pull method to agitate the embryos off of their attachment/contactsite (Fig. 2, see Note 3).

3.4. Loading the MECC

1. Using traditional embryo-handling techniques, load the embryos approximatelyhalfway down into the funnel. In general, slightly elevating the funnel end of thedevice is sufficient for embryos to pass through the funnel, into the channel, andtoward the constriction region. Gentle tapping may also be necessary (see Note 4).

2. Loaded channels must be kept at a slight incline (raise the funnel end of theMECC 4–5 mm higher than the other; this can be accomplished by resting oneend on a stack of 4–5 microscope slides) to prevent embryos from migrating backtoward the funnel during incubation.

3. Cover the medium in the inlet and outlet wells with enough mineral oil to preventevaporation.

3.5. Unloading the MECC

1. On the day of recovery, tilt the outlet well above the inlet well. The embryos willmigrate back to the funnel region into the reservoir of medium. This should bedone under a stereomicroscope to observe when the embryos reach the funnelregion.

2. Once in the funnel region, lay the MECC flat. Using traditional micropipetting,pick up the embryos before they settle back into the channel (see Note 5).

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4. Notes1. Humidity in the incubator must be close to 100% to prevent bubble formation

and minimize growth of existing bubbles. Bubbles are not problematic if chan-nels and medium are properly preequilibrated and the humidity level is correct.This requires checking the water level in the incubator on a regular basis. If prob-lems with bubbles persist, an additional small-humidified chamber may berequired to incubate the MECC.

2. Checking the microchannel for bubbles prior to embryo loading allows the bubbleto be extracted in an easier manner.

3. The push–pull method use a 1-mL syringe attached to the Luer fitting. Depend-ing on the location of the bubble before (push method) or after (pull method) theCR determines which method is used for extraction of the bubbles. The push-pullmethod needs to be done with care if the microchannel is loaded with embryos.Checking the microchannel prior to loading of embryos allows for easier bubbleextraction from the microchannel.

4. Preparation, loading, and unloading are the same for murine, porcine, and bovine.The only difference between species is the culture medium; e.g., M16 for mouse,NCSU-23 for porcine, and SOF for bovine. Gravity will help position the embryosin the desired location during loading and unloading.

5. Patience is needed to acquire each and every embryo, whether it is one or more atonce depending on the number that appear in the funnel area for retrieval.

AcknowledgmentThe authors would like to thank Kathryn Haubert of Vitae, LLC, Madison,

WI, for the photograph, illustration, and description of the MECC.

References1. Center of Disease Control and Prevention Assisted Reproductive Technology

Success Rate. National Summary and Fertility Clinic Reports. 1999, pp. 1–469.2. Choi, S. J., Glasgow I. K., Zeringue H., Beebe D. J., and Wheeler M. B. (1998)

Development of microelectromechanical systems to analyze individual mamma-lian embryos: embryo biocompatibility. Biol. Reprod. 58 (Suppl. 1), 79.

3. Glasgow, I. K., Zeringue, H. C., Beebe, D. J., Choi S. J., Lyman, J., and WheelerM. B. Individual embryo transport on a chip for a total analysis system. ThirdConference on Micro Total Analysis Systems, Banff, CA, October 13–16, 1998.

4. Chan, N. G., Lyman J. T., Choi S. J., Zeringue H. C., Glasgow I. K., Beebe D. J.,and Wheeler M. B. (1999) Development of an embryo transport and analysis sys-tem: material biocompatibility. Theriogenology 51, 234.

5. Glasgow, I. K., Zeringue, H. C., Beebe, D. J., Choi, S. J., Lyman, J. T., Chan N.G., and Wheeler, M. B. (2001) Handling individual mammalian embryos usingmicrofluidics. IEEE Trans. Biomed. Eng. 48, 570–578.

6. Raty, S., Davis, J. A., Beebe, D. J., Rodriguez-Zas, S. L., and Wheeler, M. B(2001) Culture in microchannels enhances in vitro embryonic development of pre-implantation mouse embryos. Theriogenology 55, 241.

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7. Beebe, D. J., Wheeler, M. B., Zeringue, H. C., Walters, E. M., and Raty S. (2002)Microfluidic technology for assisted reproduction. Theriogenology 57, 125–136.

8. Takahashi, Y. and First N. L. (1993) In vitro development of bovine one-cellembryos: influence of glucose, lactate, pyruvate, amino acids, and vitamins.Theriogenology 37, 963–978.

9. Petters R. M., and Wells K. D. (1993) Culture of pig embryos. J. Reprod. Fertil.48 (Suppl. 1), 61–73.