Laboratory Animal Resource Center (LARC)

Cryopreservation and Resuscitation

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RESUSCITATION AND CRYOPRESERVATION CORE:

CRYOPRESERVATION:

  • The purpose of Cryopreservation Core is to ensure:
      • Preservation
      • Protection
      • Availability
      • Sharing capacities

Cryopreservation

Sperm

Embryo

Description
  • Sperm and epididymal tissues are cryo preserved in vials
  • Stored in liquid nitrogen
  • Quality controls include motility of sperm after thawing (PI will be notified if sperm quality is unacceptable)
  • In vitro fertilization (IVF) is preferred method to obtain large number of embryos
  • IVF can be performed for embryo cryo, rederivation, resuscitation from frozen sperm, and to rapidly expand mouse colonies
  • Use 2 IVF’s per line avg.

Number of Mice Needed

  • 2 healthy male mice (reproductively active)
  • 2 to 3 males
  • Females (2 IVF's) - WT purchased by core (approx. 40-50) or provided by lab for homozygous lines
Amount Generated
  • 18-20 vials
  • Cryoarchive 200-250 embryos per line (8 straws). Number will vary with number of females provided. See note below
Age of Needed Mice
  • Males: 7-20 weeks
  • Males: 7-24 weeks
  • Females: 3-12 weeks
Time Line
  • 2-4 weeks
  • 4-8 weeks
Other Details
  • Rapid
  • Low upfront cost, higher recovery rate
  • Preserves one-half genome
  • IVF needed for sperm recovery
  • Recommended for single mutations on a common inbred background
  • Reliable
  • Higher upfront costs, lower recovery rates
  • Preserve donor genotype
  • Recovery is simple and the time is short
  • Recommended for homozygous, multiple unlinked mutations, unique genetic backgrounds

Note: The females of the same genetic background will be purchased from approved vendors for heterozygous lines. Investigators needing homozygous mice have to provide an adequate number of homozygous females, approximately 12-15 young females per IVF (3-12 weeks old). For some homozygous lines this number of females may not generate enough embryos. If more than 2 IVF’s are needed, extra charges may be incurred.

- How to request services:

  • To complete process investigator must:
    • Complete Cryopreservation request form through RIO. Indicate the original building and room location of mice, exact strain name, cage card number, DOB of each mouse and provide the required number of mice.
    • After Cryo Core receives and approves the request, then lab must submit a transfer request through RIO to PSB 528 - Attn Khalida Sabeur and indicate Cryo.
    • Make sure the strain name on the request matches the name indicated on the cage card.

Note:  All movement of mice must be coordinated through the LARC shipping office as an inter-facility transport.  Please click here for the shipping link.

Complete embryo storage records are maintained and a pdf update inventory list will be sent to the PI upon request.

For questions on services, please contact Khalida Sabeur (SabeurK@stemcell.ucsf.edu).

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SPERM OR EMBRYO RESUSCITATION:

  • Frozen sperm will be resuscitated by IVF and embryo transfer.  We will either purchase wild type oocyte donors of the same genetic background or the investigator has to provide homozygous oocyte donors of the same strain (10-12 females).
  • Frozen embryos will be recovered, cultured and transplanted into the oviduct or uterus of a pseudopregnant recipients.
  • If sperm or embryos were frozen at another institution
    • We will only receive straws or vials from mice in a specific pathogen-free (SPF) facility.
    • A thawing protocol and specific information about embryo stage and sperm quality need to be submitted.
    • A minimum of 2 straws or vials per strain should be shipped to the Cryo Core in liquid nitrogen dry shipper (please include recharge account and shipping address for return of the dry shipper).
  • Steps involved:
    1. The investigator submits a Resuscitation request form through RIO.
    2. Cryo Core reviews the completed form and approves it. Requests will not be processed if a valid recharge code is not provided.
    3. Upon receipt of the request form the Cryo Core Staff will contact you to ask to you to initiate the transfer of mice (to PSB 528) or frozen sample to the designated Cryo Preservation Core location.
    4. Cryo Core staff will update you on the date the job is scheduled and again upon completion.
    5. Cryo Core will notify you if there are any problems.

PROCEDURE RATES AND HOUSING:

Service

Cost

Time Line

Sperm Cryo No Test Thaw-culture (2 males)

$330.68

2-4 weeks

Sperm cryo and Test Thaw-culture

$789.39

2-4 weeks

Sperm Resuscitation*

$1,762.71

9-12 weeks

Embryo Cryo (2 IVF's)

$1,899.70

4-8 weeks

Embryo Resuscitation*

$1,232.87

8-10 weeks

In Vitro Fertilization

$1,881.41

8-12 weeks

Storage vials (per vial per year)

$6.26

Billed twice a year (Jan. and July)


* For resuscitation included per diem about 8 weeks.

QUALITY CONTROL PROGRAM:

  • There are different levels of quality control:
    • Sperm Cryopreservation: One vial from each strain is thawed for motility and viability.  The recovery of sperm by IVF to blastocyst stage or to live pups will be at extra charge to cover the IVF procedure.
    • Embryo Cryopreservation: A straw from the strain being cryopreserved (SW) will be cultured to determine the proportion that develop to the blastocyst stage.  In addition, the embryos (in C57BL6J) will be transferred into pseudopregnant females to produce live born offsprings.
    • Sperm and Embryos: They are properly stored in containers in LN2. The containers are correctly identified using a color-coded system and a standard format which includes strain and PI name, cryopreservation date, number and stage of embryos, and ID number.
      • The embryos will be stored in the LARC facility.
    • Records: Complete animal, sperm, and embryo records are maintained in a secure database.

CELL STORAGE:

  • The Cryo Core handles tracking, storage and inventory of frozen samples.
  • Tanks: All tanks are continuously monitored by alarm systems and undergo regular daily visual inspections.
  • Storage fees are separate from service fees noted above. 

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