Cryopreservation of cells

Samples of cells should be frozen in liquid nitrogen at all phases during the culture process, to provide a reserve in case of contamination or loss of antibody. Cells must be frozen slowly but thawed rapidly. 

Harvest approx. 10 x 10 cells from culture, and resuspend the cell pellet in 0.5 ml RPMl/FCS. 

5 ml RPMI/FCS containing 20% DMSO so that the final concentration of DMSO is 10%. 

Place the tube either in a ireezer with controlled temperature reduction, or in the neck of a liquid nitrogen Dewar, or at -SOX for 2 h. 

Transfer to rack in liquid nitrogen storage vessel. 

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T suraeva, A.A.(Ed.)1983. Cryopreservation of Cells Suspensions. Kyiv, Ukraine Naukova Dumka (in Russian). 

Cryopreservation of cells is well described (15) and covered in Chapter 2. 

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... 

Finkle BJ, ZavolaME, Ulrich JM. Cryoprotective compounds in the viable freezing of plant tissues, in Cryopreservation of Plant Cells and Organs (Kartha KK, ed.), CRC Press, Boca Raton, FL, 1985, pp. 75-113. 

Kartha KK. Cryopreservation of Plant Cells and Organs, CRC Press, Boca Raton, FL, 1985. 

Stem cell therapy involves infusion of specialized cells utilized to perform specific functions. The traditional use of cell therapy includes harvest and cryopreservation of autologous hematopoietic cells either from the bone marrow (old approach) or mobilization and pheresis of hematopoietic stem cells from peripheral blood using stem cell-mobilizing cytokines such as hematopoietic colony-stimulating factors (G-CSF, GM-CSF) or chemokine inhibitors (AMD-3100). A more recent stem cell source is umbilical cord blood that has rich pleuripotent potential and can engraft at lower doses than bone marrow or mobilized peripheral blood stem cells. 

This paper covers our recent results devoted to cryopreservation of hemopoietic stem cells from cord blood and human fetal liver. 

Therefore now the most perspective for cryopreservation of cord blood NCs is the designing of freeze-thawing methods with no washing-out using cryoprotectants, non-penetrating into a cell. 

As a result of performed by us fundamental studies there was developed a new cryopreservation method with no washing-out for cord blood NCs, it is based on use of non-penetrating PEO-1500 cryoprotectant in combination with cold treatment of cells before freezing and special own two-step freezing program. 

One more positive consequence of cryopreservation method use with PEO-1500 is noted by us higher survival after cryopreservation of hemopoietic stem CD 34 - cells in respect of NCs CD 45 - cells, that enable speaking about higher cryoresistance of the population of hemopoietic stem cells under these conditions. 

Thus, the results of present research testify to the fact that candidates to stem hemopoietic cells, obtained from two different sources human fetal liver and cord blood, demonstrate quite a high viability after cryopreservation with various methods using cryoprotectants of different effect mechanisms. Revealed differences in cryosensitivity of CD 34 cells in respect of CD 45 cells may be explained by various ratios of cells, expressing these antigens. So, in fetal liver among nucleated CD 45 - cells about 35% express CD 34 ... 

Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biocheml997 64 27S-294. 

Although particularly problematic in the Porifera, the problem of contamination, mainly by unicellular eukaryotic organisms, is an area of focus. This obstacle necessitates the validation of cell type in the culture system. Other areas in which new approaches are being sought include the selection of cell sources for the initiation of cultures and cryopreservation protocols for the maintenance of source cells for long-term studies. 

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