Vitrification as an approach to cryopreservation

Fahy, Gregory M.; MacFarlane, Douglas R.; Angell, C. Austen; Meryman, Harold T.

doi:10.1016/0011-2240(84)90079-8

Cited by 998 publications

(547 citation statements)

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“…Vitrification has been proposed as the only means of long-term protection against freezing damage (4,10) and has been found to be a natural component of freeze protection in ' Supported in part by National Institutes of Health grant GM 17959 and by National Science Foundation grant DMB 8505491.…”

mentioning

confidence: 99%

The Glassy State in Corn Embryos

Williams¹,

Leopold²

1989

Plant Physiol.

225

133

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The possibility is examined whether seeds may survive the desiccated state in part by vitrification, or the formation of a glassy state. Embryos excised from viable corn (Zea mays L.) seeds at low moisture contents show a series of low temperature first-and second-order phase transitions in the differential scanning calorimeter. These embryos produce normal seedlings if moistened. The thermal events can be duplicated almost entirely in both extracted lipids and purified commercial com oil. They are therefore associated primarily with these bulk lipids, since membrane phospholipids are present in too small an amount to produce a detectable signal. When the bulk lipids have been extracted, a glass transition appears in the remaining material. At low water contents, it occurs above +400C and systematically falls to below -600C as the water content of the embryo rises to 20%. These data are consistent with our hypothesis that the desiccated state in seeds is a glassy state, and that imbibition of water reduces the glass transition temperature below ambient, allowing biochemical activity to resume.The means by which living materials such as seeds can survive desiccation presents an interesting and difficult problem. As water is removed, the stresses imposed on membranes and other structures may be very large (19), and the maintenance of bilayer organization of membranes may be threatened (3,8). The drying of seeds will lead to enormous concentrations of solutes with an associated threat of crystallization. Yet, the moisture isotherms of orthodox seeds consistently show a region of water "binding" at low water contents (15, 16) which should be absent if substantial crystallization had occurred during drying (7).In this paper we examine the hypothesis that dry seeds exist We have studied embryos from corn seeds by differential scanning calorimetry (DSC2), seeking evidence ofglass signals. We report the existence of glass transitions both in the lipids ofthe embryos and in the nonlipid components. The evidence for glass formation as a function of temperature and water content indicates that corn embryos exist in a glassy state at room temperature when water contents are below 12% g/g dry weight. MATERIALS AND METHODSAll experiments reported were performed in a Perkin-Elmer DSC-4 differential scanning calorimeter with computer-aided data analysis ('TADS'). The thermal head is cooled with liquid nitrogen and the instrument has been modified in several particulars in order to run reliably at low temperatures (18). To obtain a flat baseline, thermal data from the calorimeter were compared with stored data obtained using empty pans ('Scanning Autozero').Unlike the more familiar first-order transitions, no heat of melting is evolved or absorbed when a glass transition is passed. The only distinctive characteristic of these secondorder transitions is a change in heat capacity; in the aqueous glasses we will discuss, this involves mostly a change in the translational mobility of the water in the sample. These changes ar...

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mentioning

confidence: 99%

The Glassy State in Corn Embryos

Williams¹,

Leopold²

1989

Plant Physiol.

225

133

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“…Cell cryopreservation can be achieved by either slow-freezing or vitrification (Coger and Toner 2000;Fahy et al 1984;Mazur 1984;Rall and Fahy 1985). The former relies on the formation of extracellular ice (the crystalized state of water) to freeze concentrate the extracellular solution in the presence of a low concentration (< ~ 2 M) of cryoprotectants (or cryoprotective agents, or CPAs for short) such as glycerol, ethylene glycol, PROH (1,2-propanediol or propylene glycol), and DMSO (dimethylsulfoxide).…”

Section: Cryopreservation At Cryogenic Temperaturesmentioning

confidence: 99%

“…In other words, no (or negligible) ice formation or freezing will occur in the sample during cooling (Fahy et al 1984;Fahy et al 2004b;Rall and Fahy 1985). Conventional vitrification (A→F→II in Fig.…”

Section: Conventional Vitrificationmentioning

confidence: 99%

“…1) has also been studied for cryopreservation of both cells and tissue. In this approach, biological samples (state A) are first loaded with a very high concentration of CPAs (up to ~ 7 M, state F) (Fahy et al 1984;Fahy et al 2004b;Rall and Fahy 1985). The samples are then cooled directly from ambient temperature to a cryogenic temperature usually in liquid nitrogen (state II) and stored there for future use.…”

Section: Conventional Vitrificationmentioning

confidence: 99%

“…However, cryopreservation of cells encapsulated in the large (≥ ~ 250 µm) microcapsules by slow-freezing has been challenging because the inevitable ice formation always results in significant damage to the microcapsules, which in turn can damage the encapsulated cells (Heng et al 2004;Herrler et al 2006;Stensvaag et al 2004;Wu et al 2007). Although the conventional vitrification approach can overcome this problem, the unusually high concentration CPA needed is detrimental to stress-sensitive cells such as the ES cells (Fahy et al 1987;Fahy et al 1984;Fahy et al 2004b;Rall and Fahy 1985;Wu et al 2007). By careful cryomicroscopy and scanning calorimetry studies, it was identified in a recent publication that water enclosed in ~100 µm (in diameter) alginate microcapsules can be preferentially vitrified over the bulk water (where the microcapsules are suspended) with only 1.4 M DMSO at a cooling rate of 100 o C/min (Zhang et al 2010).…”

Section: Conventional Vitrificationmentioning

confidence: 99%

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