Isolation (0 hr)

FIGURE 6.10 Hypothermic preservation of isolated human liver cells. Multiple enzymatic assays were performed on human liver cells hypothermically preserved in HTS-BASE supplemented with 10% fetal bovine serum (HTS-FBS), HTS-FRS, and HTS-FRS supplemented with 10% fetal bovine serum. Note that while all assays measured different functional parameters, most of the assays had a similar hierarchical order between sample types.

cryopreserved multiple times and both cell proliferation and viability do not appear to be grossly compromised as a result of these multiple preservation procedures. Yet when these cells are placed on the appropriate microporous membrane system designed so that the cells can stratify and differentiate into the engineered construct depicted in Figure 6.5, cells cryopreserved multiple times are unable to differentiate fully and form a stratified epidermis. This observation implies that multiple cell and tissue-specific functional assays should be considered carefully for human stem cells cryobanked for subsequent use in cell therapy applications.

6.3.4 Tier 4 Assays = Genomic and Proteomic Assays

Tier 4 assays consist of a group of protocols that have not yet been tested extensively by preservation biologists but may prove to be useful in the future design of improved preservation solutions and protocols. Both cDNA microarrays and protein chips are available through a number of vendors that can analyze preservation-induced stress pathways (e.g., SuperArray Bioscience) as well as examine the protein profile of cells that have been subjected to hypothermic storage or cryopreser-vation (e.g., Ciphergen). Our group has tested Ciphergen's SELDI ProteinChip system for its ability to distinguish changes in cells subjected to different cryopreservation protocols. As noted in Figure 6.11, clear differences can be noted in the profiles, yet it is unclear at this time how these data can be used to predict viability, function, or both. Yet these assays will be crucial to the advancement in preservation biology given that they have the capability of determining which of the stress pathways are activated in cells. Once known, designing improved protocols or solutions that can suppress these pathways may lead to improved procedures for cell preservation.

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