Improved cryopreservation of tissues for biological analysis
Lead Research Organisation:
University of Manchester
Department Name: School of Medical Sciences
Abstract
The PhD research will test the quality of preserved tissue by immunohistochemical and electron microscopy studies while examining the feasibility to isolate nucleic acid from laser captured material to perform next generation sequencing analysis. It will also compare the quality of readouts generated from samples processed by currently available methodology.
Importantly, the project will focus upon streamlining the overall process to enable the rapid delivery of molecular analysis that could be used for genetic screening and systems biology investigations.
Chemical fixation (e.g formalin) is a convenient method that is routinely used to preserve tissues for routine pathological analysis but is limited for more intensive study due to antigen disruption and nucleic acid fragmentation. Vitrification followed by freeze substitution represents the current gold standard preparative method for the high-resolution imaging of biological materials and also maintains nucleic acid integrity for the range of molecular analytical methods including next generation sequencing.
However, current equipment for vitrification is large, expensive and requires skilled operators. In this project, we will develop Progressive Lowering of Temperature (PLT) techniques to produce samples that maintain good ultrastructure in the frozen state with high cell viability upon thawing.
Importantly, the development of the Stirling Cryocooler platform by Asymptote provides opportunity to develop PLT capability that would obviate the requirement for expensive process equipment currently used to vitrify samples by rapid cooling. Upon validation of the PLT process, cryopreserved tissue samples will be subjected to multiple analyses.
The PhD seeks to address a developing issue relating to the preservation of tissue for high intensity molecular and cellular analysis. To this end, this project bridges physics, material science and molecular biology to provide a broad training opportunity.
Importantly, the project will focus upon streamlining the overall process to enable the rapid delivery of molecular analysis that could be used for genetic screening and systems biology investigations.
Chemical fixation (e.g formalin) is a convenient method that is routinely used to preserve tissues for routine pathological analysis but is limited for more intensive study due to antigen disruption and nucleic acid fragmentation. Vitrification followed by freeze substitution represents the current gold standard preparative method for the high-resolution imaging of biological materials and also maintains nucleic acid integrity for the range of molecular analytical methods including next generation sequencing.
However, current equipment for vitrification is large, expensive and requires skilled operators. In this project, we will develop Progressive Lowering of Temperature (PLT) techniques to produce samples that maintain good ultrastructure in the frozen state with high cell viability upon thawing.
Importantly, the development of the Stirling Cryocooler platform by Asymptote provides opportunity to develop PLT capability that would obviate the requirement for expensive process equipment currently used to vitrify samples by rapid cooling. Upon validation of the PLT process, cryopreserved tissue samples will be subjected to multiple analyses.
The PhD seeks to address a developing issue relating to the preservation of tissue for high intensity molecular and cellular analysis. To this end, this project bridges physics, material science and molecular biology to provide a broad training opportunity.
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M016463/1 | 01/10/2015 | 30/06/2020 | |||
| 1640493 | Studentship | BB/M016463/1 | 01/10/2015 | 30/09/2019 | Adam Milner |