Embryo and Sperm Cryopreserve

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Resources and Recommendations

Embryo Cryopreservation: For strain cryopreservation, 10-12 homozygous or heterozygous stud males will be mated with superovulated wildtype females. Approximately 250-300 eight cell stage embryos will be collected and frozen. Roughly 20-30 embryos will be stored per straw, which can then be stored in liquid nitrogen in the Core’s freezers. Freezing of 8-cell embryos is the most stable stage for embryo cryopreservation and storage.

Sperm Cryopreservation: This is the most fast and least expensive option for cryopreservation of mouse strains. Frozen sperm can be stored in LN2 indefinitely, and reconstitution of live mice can generally be achieved using IVF. Following collection of epididymal sperm, 20 samples will be frozen and stored in liquid nitrogen. However, keep in mind that IVF is inefficient with frozen-thawed sperm from some strains such as C57BL/6, which has critical implications since many induced mutant strains are maintained as congenic strains on that background. Therefore for greater reliability during recovery, we recommend using our Speed Cryo service (see below) for freezing a strain on the C57BL/6 inbred background.

IVF embryo cryopreservation (Speed Cryo): The freshly collected sperm from 3-4 transgenic males is used to fertilize the eggs of superovulated females of the same strain background via in vitro and then cryopreservation of 2 cell or 6-8 cell stage embryos is done. Simultaneously we also freeze 10 samples of sperm from these males. Thus, your strain will be cryopreserved and stored as both embryos and as well as sperm.

Despite several approaches of cryopreservation exist we recommend to employ Speed Cryo as for C57BL/6 and as well as for other mouse lines.

Intracytoplasmic sperm injection (ICSI): Currently we are in process establishing ICSI of frozen sperm into unfertilized oocytes.

Quality Control: Each strain frozen by standard method or Speed Cryo can be checked to ensure overall quality and viability of embryos using one of two methods:

  1. Following standard or Speed Cryo freezing, one straw will be thawed, and the embryos will be placed in culture for development to blastocysts where a >75% development to blastocysts will be considered a successful cryopreservation.
  2. Following standard or Speed Cryo freezing, one straw will be thawed, and the embryos will be placed in culture for development to blastocysts followed by a uterine transfer into a pseudopregnant recipient. The generation of live pups displaying the appropriate genotype (genotyping to be done by the customer) will be considered a successful cryopreservation.
  3. After sperm cryopreservation one straw will be thawed and a quality of the spermatozoa will be visually evaluated under a microscope.

Long-term storage of Germplasm: The frozen embryos and sperms are stored in liquid nitrogen in two freezers located at two geographically distinct sites: one located on the OHSU Main Campus and the other at the West Campus. Each freezer is monitored by OHSU Public Safety using remote alarm.

Responsible Parties and Timeline

Cryopreservation Timeline

Service ordering

To initiate a freezing project, please fill out and forward a Cryopreservation Request Form to the TMM. In addition to the Project Request Form, please provide the following materials:

  • For standard cryopreservation of 8-cell stage embryos provide 10-12 stud males at the age of 2-9 months.
  • For Speed Cryo provide 3-4 stud males at the age of 2-9 months. For Sperm Cryopreservation provide 3-4 stud males at the age of 3-9 months.
Standard Embryo cryopreservation service fees include:
  1. Purchase of virgin C57BL6/J or B6xDBA-2F1 females (donors of oocytes).
  2. Cryopreservation of 250-300 eight -cell embryos.
  3. Cryostorage of frozen embryos for 1 month.
Speed Cryo cryopreservation service fees include:
  1. Purchase of virgin C57BL6/J or B6xDBA-2F1 females (donors of oocytes).
  2. Cryopreservation of 250-300 eight cell embryos.
  3. Cryopreservation of 10 samples of sperm.
  4. Cryostorage of frozen embryos and sperm for 1 month.
Sperm cryopreservation service fees include:
  1. Cryopreservation of 20 samples of sperm.
  2. Cryostorage of frozen sperm 1 month.
Service fees do not include:
  1. Test thaws of control embryos.
  2. Test thaws Speed Cryo frozen embryos.
  3. Test thaws of frozen sperm and IVF.
  4. Transfer of embryos into pseudopregnant recipient females.
  5. Control of frozen germplasm. This is very important, therefore we recommend to order, and pay the small additional fees.
  6. Long term storage of embryos and sperm. 
  7. Shipping charges.
Additional Service

According to customers’ request, TMM can purchase and use other mouse lines for the cryopreservation of genetically modified mice. These additional services will be charged further fees, including the extra costs of the cage(s) per diem.

Service fees

See Fees, Cryopreservation.