Long-term biopreservation of cells and tissues has a broad impact in multiple fields including tissue engineering, regenerative medicine, stem cells, blood banking, animal strain preservation (biodiversity protection), clinical sample storage, transplantation medicine and in vitro drug testing. Vitrification (ice/crystal-free cryopreservatio) has emerged as a novel approach over traditional slow freezing methods. Although vitrification minimizes mechanical damage due to ice crystal nucleation, it suffers from toxicity due to high concentrations of cryoprotectant agents (CPAs). The current vitrification methods require extremely high levels of CPAs of up to 8.2 M that are cytotoxic and cause osmotic shock. Also, the lengthy manual processing steps of current vitrification methods add to the technical complexity, require highly trained technicians, and result in variations between users. For instance, low CPA-level vitrification has immense potential for the stem cells compared to other methods in preserving their functionality. Recently, we demonstrated that we can achieve vitrification at ultra-rapid freezing and thawing rates with as low as 1.5M CPA concentration. We are adapting this new knowledge to the vital needs of cell cryopreservation at the clinic including discarded anonymous human oocytes. This proposal investigates a new experimental strategy to minimize the CPA concentrations and improve clinical outcomes using novel technologies (i.e., nanoliter droplet vitrification). These steps are facilitated by theoretical understanding o the underlying mechanisms governing vitrification. The expected outcome of this study is a closed-system platform technology with broad applications to human cell (e.g., hepatocytes, oocytes, sperm, stem cells), tissues (e.g., blood), micro-tissues (e.g., embryoid bodies, islets) covering areas of reproductive medicine, tissue engineering and regenerative medicine as well as to wild life preservation. These studies can also significantly impact the care of infertile couples and facilitate fertility preservation.

Public Health Relevance

Long-term biopreservation of cells and tissues has a broad impact in multiple fields including tissue engineering, regenerative medicine, blood banking, wild life preservation (biodiversity protection), clinical sample storage, transplantation medicine, reproductive medicine and in vitro drug testing. This proposal creates a novel technology platform and a new strategy to minimize the CPA concentrations in vitrification (ice/crystal-free cryopreservation) and to improve clinical outcomes using novel nanoliter droplet technologies, significantly impacting the care of infertile couples and facilitating fertility preservation. The expected outcome of this study is a closed-system platform cryopreservation technology with broad applications to human cells (including oocytes, sperm, and stem cells), tissues (including blood), micro-tissues (including embryoid bodies, islets).

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB015776-04
Application #
8686838
Study Section
Instrumentation and Systems Development Study Section (ISD)
Program Officer
Hunziker, Rosemarie
Project Start
2012-08-01
Project End
2016-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
4
Fiscal Year
2014
Total Cost
$385,463
Indirect Cost
$125,018
Name
Stanford University
Department
Radiation-Diagnostic/Oncology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
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