Cryopreservation of stem cells

Stem cells are special type of cells which can renew themselves, and can grow into many other cell types. Scientists have found stem cells not only in embryo (so called embryonic stem cells) but also in adults (adult stem cells, e.g from bone marrow). Stem cells could be very useful for tissue/organ repair (e.g brain, heart and other tissue). But in order to apply stem cell based therapies into clinic settings, many technical obstacles must be overcome. One of these is how to preserve stem cells for long term. The most feasible method is cryopreservation. For example, when a baby is born, the stem cells in the cord blood can be collected, separated, frozen and stored at liquid nitrogen temperature (-196 oC). If in later stage in his (or her) life the individual needs stem cells, his (or her) cells can be thawed and (appropriate) therapy can be started. Other types of stem cells can similarly be preserved. The problem is that stem cells do not like low temperatures (not a surprise). They may simply die, or lose some important functions. The additional complexity is that it is not easy to tell what/how many functions are lost. This project focuses on two important questions (1) how to make sure stem cell functions be preserved by using better cryopreservation agents (e.g use of antfreeze proteins protecting fishes in arctic) and thereby adjusting how fast we freeze the cells, and (2) how to detect any damage by using new type of microscope to three dimensionally see inside and around the cells. At the end of the project, we hope we will have optimal cryoprotecting chemical formulation and optical protocols, which can be used by stem cell banks, research laboratories, hospitals and industry.

Cryopreservation is the process of choice for long term preservation of stem cells but the process imposes extreme conditions on stem cells leading to functional loss or even cell death. Cryopreservation is a sequential process consisting of addition of cryoprotecting agents (CPA), freezing before storage and thawing and removal of CPA, before use. Hence each step ( and the overall process) must be optimised to minimise functional loss. The proposed project will address two interrelated aspects; firstly how to minimize the damage to stem cells during cryopreservation and secondly how to assess the damage to stem cells quickly and non-destructively. In this project, we propose to test different CPA formulations (conventional CPA, macromolecules, antifreeze proteins, antioxidants) at different concentrations, addition and removal methods, freezing and thawing rates on stem cell viability and functions, assayed by proliferation, differentiation , surface markers and gene expression. We will develop novel detection methods based on near infrared (NIR) nonlinear optical microscopy (NLOM) for rapid, non-invasive and non-destructive functional assays on the possible functional loss of the cryopreserved stem cells, with the aim to reduce/replace the laborious and time consuming and costly assays. In this proposal, we plan to use monoclonal mouse embryonic , mouse and human mesenchymal stem cells , and human haemotepoetic stem cells from umbilical cord blood, but the developed protocols and methodologies can be readily extended to other types of stem cells.