Cryopreservation techniques

The recent development and application of methodologies sensitive at the single cell wall level has shown that traditional bulk analytical techniques average out important intrinsic heterogeneity in sampled populations. By exploring the diversity of cell walls using novel cryopreservation techniques for electron microscopy and non-invasive... 

Since Harrison, in 1907, first microscopically observed neurite outgrowth from cultured frog neurons, many have experienced the powerful persuasion of direct visualization of cellular responses in culture. Standardization of media composition, introduction of antibiotics, and development of cryopreservation techniques and immortalized cell lines in the 1950s enabled routine application of cell culture in the biological research laboratory. Utilization of cell culture in toxicology soon followed with development of systems for monitoring chemically induced mammalian genotoxicity. 

The cryopreservation technique has been established for some cells7 As a result, these cryopreserved cells have been used in many studies. However, in the case of neurons, the appropriate cryopreservation process has not yet been established because of their complex structure, low mitotic activity, and uncertainties regarding the cryoprotection mechanism. Thus, primary culture cells are used for studying neurons. 

Recent development in cryopreservation techniques partially solved the problem with accessibility of many cell types, in particular primary cells. On the other hand, it raises additional concerns regarding phenotype and functionality of cells being exposed to freeze-thaw cycle. Therefore, assessing cell recovery, viability, and functionality after thawing is deemed to be essential. Ideally, the acceptance criteria to be met before using such a system should be established up front and monitored continuously. Moreover, since high concentrations of DMSO are often used as the cryopreserva-tive agent, its effect on the functionality of the test system should be considered. If necessary, additional incubation steps to reduce such an effect are recommended. 

A number of commercially available sources of human liver cytochrome enzymes exist (see above). There are deficiencies or drawbacks associated with many of them. Most sources of primary hepatocytes provide cells which are notoriously difficult to culture for any significant length of time in the laboratory without a loss of cytochrome phenotype. Cryopreservation techniques to prolong the storage lifetime of particular hepatocytes isolates also routi-... 

Because the success of any given cycle of IVF treatments is by no means guaranteed, many couples choose to use cryopreservation techniques to freeze embryos produced in one cycle for future use. This streamlines the process a couple must go through if treatment does, in feet, have to be repeated. It also reduces the need for performing invasive procedures on the female patient. 

With the advent of cryopreservation techniques, it is now possible to preserve the arterial endothelium and smooth muscle cells and their properties of contractility and vessel wall relaxation (42,43). A large retrospective multicenter study examined a population in which either fresh or cryopreserved arterial allograft was used for limb salvage infrainguinal reconstruction. The data from this report demonstrated suboptimal 5-year primary and secondary patency rates of 16% and 26%, but a respectable 74% 5-year rate of limb salvage (44). While it does have drawbacks, the use of arterial allograft, either fresh or cryopreserved, represents another available conduit with which to allow reconstruction for limb salvage. 

The supply of living cestode material for in vitro and in vivo experiments presents a major barrier to progress in research. It is therefore worth drawing attention to the fact that recent work by Eckert Ramp (191) have shown that cystic Echinococcus mutilocularis can be maintained successfully by cryopreservation without the proliferative capacity being lost. The establishment of this most useful technique should act as a great stimulus to future work. For technical details, the original paper (191) should be consulted. 

The collection of plant tissue is quite different from animal tissue collection. The discussion of collection of plant and animal tissue by Dessauer et al.2S is detailed and helpful. However, the recommendations for procedures unique to plant tissue collection are somewhat misleading and outdated, especially when tropical collections are involved. Plant tissue can now be collected and transported as either fresh tissue (leaves and/or shoot cuttings) or preserved tissue the latter either as cryopreserved tissue (liquid nitrogen or dry ice) or as dried tissue (air-dried, herbarium-dried, lyophilized, or chemically dried). Ambient-temperature liquid chemical preservation techniques (such as those routinely done for herbarium plant specimens in the tropics) so far have been ineffective in maintaining adequate yields of high-quality DNA.15 It should be stressed again that the manner of collecting plant tissue is dictated by several other factors what macromolecule (DNA, RNA, or isozymes) will be examined, what type of nucleic acid extraction method will be used (or, more impor-... 

Microtiter Plate Technique for the Measurement of Glutathione in Fresh and Cryopreserved Lymphoblasts Using the Enzyme Recycling Method... 

Gray RGF, Ryan D, Green A. The cryopreservation of skin biopsies— a technique for reducing workload in a cell culture laboratory. Ann CUn Biochem 1995 32 190-2. 

There are several ways to purify a primary RPE culture.88,96-100 The process of isolation, culture technique, and cryopreservation is well documented.98,99 Grown on porous supports, RPE cells have been applied in a limited number of permeability and toxicity studies.88 The isolation and culture technique of primary RVE cells from Macaca monkeys is also documented 101 however, their application in drug permeability studies has been minimal.88... 

Cryopreservation of hepatocytes allows their storage and transport rather than immediate use after isolation. However there is an associated loss of phenotype with this technique, making cryopreserved hepatocytes less physiologically relevant than primary human hepatocytes (Terry et al., 2006). 

DAY j G and BRAND J J (2005) Cryopreservation methods for maintaining microalgal cultures, in Andersen R A (ed). Algal Culturing Techniques. New York Academic Press, 165-188. 

---