Cryopreservation procedures

Care must be taken not to overtrypsinize the cells when removing them from the tissue culture surface. The 2.2.15 cells are sticky and have a natural tendency to clump. Aggregated cells do not grow well and are difficult to count. The 2.2.15 cells also perform better in reseeding and cryopreservation procedures if they receive fresh medium 24 h before trypsinization. 

Hundreds of references to sperm cryopreservation in aquatic species are available today, and this number grows each month. Many authors summarized published methods in critical reviews (see Table 3.1). Anyone attempting sperm cryopreservation for the first time is referred to these reviews as a starting point. They will find ample information on the procedure and the factors which should be considered in a given species. In this section, we will focus on the items most common in all aquatic species, and describe the steps of a cryopreservation procedure (Fig. 3.2). 

Career opportunities are extremely limited in cryonics. The handful of cryonics facilities currendy in existence employs a small staff of technicians to maintain the cryopreservation tanks and sales personnel. During a cryopreservation procedure, the staff expands tremendously for a brief period and includes physicians, nurses, and technicians. All temporary employees have full-time positions elsewhere in their areas of expertise. 

Ashwood-Smith, M.J., Genetic damage is not produced by normal cryopreservation procedures involving either glycerol or dimethyl sulfoxide a cautionary note, however, on possible effects of dimethyl sulfoxide. Cryobiology, 22,427,1985. 

Steponkus, P.L., Caldwell, S. (1993). An optimized procedure for the cryopreservation of Drosophila melanogaster embryos. Cryo-Letters 14, 375-380. 

Typical experimental procedures are as follows The test drug candidate is incubated with pooled human liver microsomes (e.g., 1 mg protein/mL) that were previously preincubated with ABT (1 or 2 mM) for 30 minutes at (37 1)°C in the presence of an NADPH-generating system. Incubations of the drug candidate in the absence of ABT serve as controls. For hepatocytes, suspensions of freshly isolated or cryopreserved hepatocytes (lx 106 cells/ mL) are preincubated with 100-pM ABT for 30 minutes in 0.25 mL of Krebs-Henseleit buffer or Waymouth s medium (without phenol red) supplemented with FBS (4.5%), insulin (5.6 pg/mL), glutamine (3.6 mM), sodium pyruvate (4.5 mM), and dexamethasone (0.9 pM) at the final concentrations indicated. After the preincubation, the drug candidate is added to the incubation and the rate of metabolism of the drug candidate is compared in hepatocytes or microsomes with and without ABT treatment. A marked difference in metabolism caused by ABT is evidence that CYP plays a prominent role in the metabolism of the drug candidate. 

Once it is possible to expand a clinically defined and safe cell lineage for transplant, it is also necessary to be able to store the viable cells for subsequent attempts or procedures. This can avoid further surgery on the same patient. This demands the development of crypreservation methods for cells and tissues, involving a tissue bank (blood, skin, bones, cornea, bone marrow, umbilical cord blood, etc.). Cryopreservation must be efficient for long periods of storage, since the frozen cells may be required years after the initial deposit. 

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-... 

These cells were cryopreserved and fresh aliquots unfrozen approximately every 6 weeks. By this procedure resistance was maintained and reproducibility of experiments over many months was assured ( ). 

Cryopreserved human hepatocytes from three male and two female donors or freshly isolated male rat hepatocytes are analyzed for viabilities (75-85%) using the trypan blue exclusion methods. Incubations are performed by suspending the hepatocytes in Krebs-biocarbonate buffer followed by addition of a H-labeled compound in methanol. The specific radioactivity of the compounds is 100 Ci/ mol. The final concentration of test compound in the suspension is 10 xM in a final volume of 1 mL (1x10 cells/mL), and the final concentration of methanol does not exceed 0.2% (v/v). Incubations are allowed to proceed at 37 °C for 1 h, and are quenched with acetonitrile (5 mL). The remaining procedures are the same as described in Section 14.2.2 (the protocol for in vitro covalent protein binding in human or rat liver microsomes a test-tube method). Covalent protein binding values in pmol-equiv./mg protein are estimated based on the residual radioactivity in the protein pellets. 

This procedure is used for cryopreservation of 8-32-cell morula stage embryos (14). Mouse cleavage-stage embryos can be harvested from the oviducts of 2-3-d pc pregnant mothers or generated by culturing fertilized one-celled embryos in Ml6 medium for 2-3 d. 

The book by Cabrita et al. (2008b) gives more detailed information on specific procedures for sperm and egg cryopreservation in marine and freshwater species. 

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. 

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