Source: CRYOCRATE LLC submitted to
A HIGHLY EFFICIENT VITRIFICATION DEVICE FOR ANIMAL EMBRYOS AND SPERMATOZOA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
1015827
Grant No.
2018-33610-28253
Cumulative Award Amt.
$99,686.00
Proposal No.
2018-00489
Multistate No.
(N/A)
Project Start Date
Jul 1, 2018
Project End Date
Feb 29, 2020
Grant Year
2018
Program Code
[8.3]- Animal Production & Protection
Project Director
Han, X.
Recipient Organization
CRYOCRATE LLC
1601 S PROVIDENCE
COLUMBIA,MO 65211
Performing Department
(N/A)
Non Technical Summary
This SBIR Phase I project aims to develop a novel ultrafast cooling device and companion cell suspension processing device that significantly reduce the need for cryoprotectants and improve the post-thaw viability and functionalities of livestock semen and embryos. These products will potentially greatly facilitate application of livestock artificial insemination (AI) and embryo transfer (ET) technologies.
Animal Health Component
40%
Research Effort Categories
Basic
10%
Applied
40%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30135992020100%
Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3599 - Swine, general/other;

Field Of Science
2020 - Engineering;
Goals / Objectives
This proposal focuses on establishment of an efficient vitrification technology using lowconcentration of cryoprotectants for livestock semen and embryos, by developing a novelultrafast cooling device and companion cell suspension processing device. These productswill greatly facilitate application of livestock artificial insemination (AI) and embryo transfer(ET) technologies. The envisaged ultra-fast cooling device can be used for both semen andembryo cryopreservation using our novel flattened straw package to achieve these requiredultra-fast cooling rates. As such, the process is directly relevant to agricultural manufacturingtechnology. The proposed method significantly lowers the cryoprotectant concentrationrequired for pig embryo cryopreservation, and simplifies the cooling process for pigspermatozoa. Increasing the throughput of both AI and ET technologies through our technicalinnovations will benefit current practices within the livestock industry and improve breedingefficiency which is a rate limiting step in productivity, and that is vitally important for promotingquality and quantity of food production worldwide that will be required to meet the challengesgenerated by world population increase and climate change. Its far-reaching influence shouldalso help solve societal issues involved in food safety and childhood obesity.
Project Methods
We will first design and produce the prototype of theproposed device system comprising an ultrafast cooling device and a cell suspensionprocessing device, test its thermal performance, and optimize its functionality throughan iterative engineering approach prior to beginning in-vitro studies. We will then achieve vitrification of pig embryos and spermatozoa using the prototypeof the proposed device system. We will perform both in-vivo and in-vitro studies to evaluatethe cryopreservation efficiency of the produced device and the proposed protocol, andcompare results obtained to those from traditional approach. The design of theprototype will be further improved based such comparisons.

Progress 07/01/18 to 02/29/20

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Using the results from the thermal studies and in vitro studies, an international patent filing was made as "Improved Ultra-Fast Cooling System and Methods of Use", PCT/US2019/26162, published as WO2019195791A1. The USPTO written opinion was that all claims are novel, involve inventive step, and industrially applicable. The PCT filing was being nationalized in USA, EU, Japan, China and India in 2021. The PI of this project is the leading inventor and the company owns the IP. CryoCrate was invited to presented the ultra-fast cooling technology and other CryoCrate technologies in Life Science Summit 2020, one of the twenty emerging company showcases, see: https://lifesciencessummit.org/presenters-20/. CryoCrate trademarked the technology as INSTAvitria®, and USPTO has granted us the trademark. CryoCrate published the associated technical details and applications on CryoCrate webpages What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Our overall goal is to develop a novel ultra-fast cooling device to improve the efficiency of cryopreservation of animal embryos and spermatozoa. With the support of this SBIR award, we first designed and manufactured the proposed device system and tested the thermal performance and systematic operation, as proposed in Aim 1 and detailed in the Interim Technical Report: we designed and manufactured a novel two-phase, blade-jet-array device to achieve the desired ultra-fast cooling rate on a surface area that is significantly larger than that demonstrated in our preliminary results using a co-axial single jet device. We also designed and manufactured a cell suspension processing device that processes cell suspensions into ultrathin straws or ultrathin layers with large surface areas to be cooled by the liquid nitrogen jet arrays of the above ultra-fast cooling device. The device also forms closed systems that prevent contamination from liquid nitrogen. We tested the thermal performance of the proposed system composing two devices. We used the device system developed from the Aim 1 work to vitrify pig embryos and semen suspensions. We first determined the post-thaw viability of intact pig embryos and spermatozoa using standard in vitro assays. After confirming the success in vitrification of intact pig embryos (25% post-thaw re-expansion, which was five times more than the control group using traditional vitrification approach without delipidation), embryo transfers were performed on two sows, using 180 and 204 embryos vitrified by our device system, respectively. One of the two sows stopped menstruation and showed a wet oviduct. Approach and Results: In the Aim 1 work, 08/01/2018 to 02/28/2019, we successfully upgraded the proposed system through an iterative engineering approach combining theoretical analyses, numerical simulation and thermal performance measurement. A new design of cell suspension processing device was made and the devices were manufactured based on the new design. The thermal performance was tested and the systematic operation efficiency evaluated. In the Aim 2 work, 03/01/2019 to 02/29/2020, we performed standard in vitro or in vivo assays of post-thaw semen and embryos. The post-thaw re-expansion rate was over 25% for intact pig embryos, which was significant improvement of traditional approach of pig embryo vitrification without delipidation (less than 10% as re-expansion rate). However, in vivo ET experiments on two sows were not successful, due to technical reasons. We didn't observe any survival from thawed semen.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: https://lifesciencessummit.org/presenters-20/, see company CryoCrate


Progress 07/01/18 to 06/30/19

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Patent publication, conference presentation, our website: Using the results from the thermal studies and in vitro studies, an international patent filing was made as "Improved Ultra-Fast Cooling System and Methods of Use", PCT/US2019/26162, published as WO2019195791A1. The USPTO written opinion was that all claims are novel, involve inventive step, and industrially applicable. The PCT filing was being nationalized in USA, EU, Japan, China and India in 2021. The PI of this project is the leading inventor and the company owns the IP. CryoCrate was invited to presented the ultra-fast cooling technology and other CryoCrate technologies in Life Science Summit 2020, one of the twenty emerging company showcases, see: https://lifesciencessummit.org/presenters-20/. CryoCrate trademarked the technology as INSTAvitria®, and USPTO has granted us the trademark. CryoCrate published the associated technical details and applications on CryoCrate webpages: What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Our overall goal is to develop a novel ultra-fast cooling device to improve the efficiency of cryopreservation of animal embryos and spermatozoa. With the support of this SBIR award, we first designed and manufactured the proposed device system and tested the thermal performance and systematic operation, as proposed in Aim 1 and detailed in the Interim Technical Report: we designed and manufactured a novel two-phase, blade-jet-array device to achieve the desired ultra-fast cooling rate on a surface area that is significantly larger than that demonstrated in our preliminary results using a co-axial single jet device. We also designed and manufactured a cell suspension processing device that processes cell suspensions into ultrathin straws or ultrathin layers with large surface areas to be cooled by the liquid nitrogen jet arrays of the above ultra-fast cooling device. The device also forms closed systems that prevent contamination from liquid nitrogen. We tested the thermal performance of the proposed system composing two devices. We used the device system developed from the Aim 1 work to vitrify pig embryos and semen suspensions. We first determined the post-thaw viability of intact pig embryos and spermatozoa using standard in vitro assays. After confirming the success in vitrification of intact pig embryos (25% post-thaw re-expansion, which was five times more than the control group using traditional vitrification approach without delipidation), embryo transfers were performed on two sows, using 180 and 204 embryos vitrified by our device system, respectively. One of the two sows stopped menstruation and showed a wet oviduct.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: https://lifesciencessummit.org/presenters-20/