We have funded the development of a microfluidic device that improves the developmental competence of in vitro derived mouse embryos to allow the use of non-surgical embryo transfer (NSET) in the generation of transgenic mice.
Challenge Contractor: Dr Virginia Pensabene
Organisation: University of Leeds
Start date: 2016
Duration: 1 year
Sponsor: MRC Harwell
In 2018, 1.49M procedures were carried out in Great Britain for the creation and breeding of genetically altered mice. Generation of these mouse models involves the transfer of genetically altered embryos into pseudo-pregnant mice. Most transfers are done surgically via a laparotomy which can cause the mice pain. NSET avoids the need for surgery but is only optimised for the transfer of late stage pre-implantation embryos (i.e. blastocysts). This makes it unsuitable for earlier embryonic stages generated through in vitro fertilisation (IVF) and pronuclear injection methods, both of which are necessary to create transgenic archives or to manipulate the embryo using CRISPR/Cas9 gene editing.
Addressing the animal welfare concerns associated with surgical embryo transfer, MRC Harwell posed the EASE Challenge to maximise the use of NSET by developing a reliable system for culturing in vitro manipulated embryos through to the blastocyst stage.
The EASE Challenge was awarded to a team, with expertise in microfluidic technology and reproductive biology, led by Dr Virginia Pensabene.
The team developed a microfluidic device that reliably permits the culture of embryos to the blastocyst stage – the technology is cheap, transparent and compatible with standard laboratory equipment. Up to 15 embryos can be cultured in the same device although there is the potential to increase this number.
3Rs and scientific benefits
In this year-long project, the Leeds team and their colleagues at MRC Harwell undertook studies to evaluate the success rate of culturing IVF embryos in the device and to test their subsequent viability (i.e. number of pups born/ embryos implanted) when transferred surgically. This demonstrated that there was a high success rate for morula/blastocyst formation from fresh and cryopreserved two-cell IVF embryos and that birth rates of more than 50% could be achieved – consistent with that achieved with IVF blastocysts formed using traditional micro-drop culture.
The number of pups born using morula/ blastocysts cultured in the microfluidic device and implanted by NSET was variable. Although pilot experiments showed that birth rates better than those with conventional culture and surgical transfer could be achieved, there is more work to be done to optimise the NSET as part of the collaboration. Ultimately this could replace 90% of the 2,500 surgical embryo transfers performed at MRC Harwell each year. More broadly, given the predicted growth in the use of gene editing approaches, tens of thousands of surgeries could be avoided worldwide by the ability to enhance the formation of blastocysts in vitro.
The microfluidic device reduces the exposure of embryos to in vitro stressors and improves their viability through to the blastocyst stage by manipulating the volume and composition of the medium, pH and oxygen tension. A functional prototype has been developed and two patent applications have been submitted in the UK to protect the technology. Virginia is in the process of setting up a spin-out company to commercialise the technology.
The device has many applications in developmental biology and to help exploit this Virginia has received £50k from the EPSRC Impact Acceleration Account to make the device compatible with time lapse microscopy. The Leeds team has already used the device in studies to understand the role of endometrial cells in embryo development, and to test the embryo toxicity of polydimethylsiloxane, a silicone used in a variety of products including medicines and cosmetics.
There are also potential non-research applications for the device. This includes clinical and agricultural applications where techniques for IVF and in vitro production of embryos are sub-optimal. IVF is an invasive and expensive treatment. There are around 60,000 fertility treatments in the UK each year, with birth rates ranging from 2% to 30% (per embryo transferred) depending on the woman’s age. In agriculture, IVF is increasingly used in cattle breeding to improve genetic selection. There is a significant market opportunity and Virginia has been awarded £80k from Grow Med Tech (a consortium of six universities across the Leeds and Sheffield areas) to modify the device so that it is suitable for human embryo culture, and £100k from the MRC’s Confidence in Concept scheme to scale-up the system for bovine embryo culture.
Sponsor in-kind contributions
MRC Harwell provided a range of in-kind contributions. This included fresh and frozen one and two-cell embryos for validation and optimisation work, and mice as embryo recipients. MRC Harwell also provided 0.2 of a full-time equivalent (technical staff).
This case study was published in our 2019 CRACK IT Review.