Principles and practical issues for cryopreservation of nerve cells.

Nerve cells isolated from the brain have a number of research and clinical applications, not the least of which is their transplantation to patients with Parkinson's disease. Neural primary and precursor cells of several areas of the brain are potential candidates for transplantation and research. However, supply of suitable tissue is one of the major problems associated with the widespread application of such techniques. The ability to store such tissue for prolonged periods would greatly alleviate this problem. Cryopreservation allows indefinite storage, provided the storage temperature is sufficiently low. Whilst many of the potentially usable cell types have been shown to be capable of surviving cryopreservation to some degree, survival post-thaw needs to be considerably improved. Cryopreservation techniques applied to date are mostly crude and often adopted from those used for unrelated cell types. Studies involving cryopreservation of primary neural cells and stem cells are reviewed, the basic principles of cryopreservation explained and suggestions made for improvements to the low temperature storage of these cells.

Cryopreservation of mammalian embryos.

The cryopreservation of mammalian embryos has expanded over the past 20 yr by encompassing a range of sophisticated methods to deal with different developmental stages and different sensitivities to low-temperature exposure. We have described a method for slow, controlled-rate freezing of early stage embryos based on exposure to 1,2-propanediol and sucrose, while the method for late-stage (blastocyst) embryos employs mixtures of glycerol and sucrose. Both methods have been used for animal and human embryos. A third rapid cooling or "vitrification" technique is described, which depends on brief but controlled exposure of multicellular embryos to mixtures of glycerol and 1,2-propanediol at high concentrations. This technique is used for successful animal embryo cryopreservation but is not yet widely applied in the clinic.

Cryopreservation of mammalian oocytes.

Two methods for the cryopreservation of mammalian oocytes are described. One method uses a relatively low concentration of the cryoprotectant propanediol plus sucrose and requires controlled-rate cooling equipment to achieve a slow cooling rate. Such a method has produced live births from cryopreserved human oocytes. The second method described employs a high concentration of the cryoprotectant dimethyl sulfoxide plus a low concentration of polyethylene glycol. This method involves cooling by plunging standard straws into liquid nitrogen vapor, hence avoiding the need for specialized equipment, but requires technical ability to manipulate the oocytes quickly in the highly concentrated cryoprotectant solutions. Murine oocytes vitrified, using this technique, have resulted in live births.

Contrast agent bubble and erythrocyte behavior in a 1.5-MHz standing ultrasound wave.

Human erythrocytes and Optison contrast agent have been exposed to ultrasound, both alone and in combination, in a single-half-wavelength chamber driven at its resonance frequency (fo) of 1.5 MHz. Cell movements were recorded by video microscopy at speeds up to 500 frames/s. The hypothesis that cells near a standing wave pressure node might be stressed by the microbubble products of sonicated contrast agent was examined. In the absence of contrast agent, cells moved rapidly to form an aggregate in the standing wave pressure node plane. First subharmonic and second harmonic emissions were detected from cell-contrast agent suspensions immediately on exposure to a threshold peak pressure amplitude of 0.98 MPa. Emissions at 3fo/2 occurred at 1.47 MPa, whereas white noise and lower-order subharmonic emissions coincided with the appearance of visible bubbles at a threshold of approximately 1.96 MPa. Cells exposed together with contrast agent at a pressure of 0.98 MPa precessed very rapidly about the pressure node plane. This behavior was discussed in the context of a recent analysis predicting that, in contrast to the situation for lower-pressure amplitudes, subresonant size bubbles translate about pressure node plane if the driving pressure amplitude is sufficiently high. Many precessing erythrocytes were clearly spiculated and this morphology persisted after the cells had left the area of precession. Hemoglobin release was significant under conditions inducing precession with first subharmonic and first harmonic emissions. Protein release increased discontinuously near the pressure thresholds, where more complex categories of frequency emission were detected. The potential of this system, which induces erythrocyte morphology changes and some protein release at the first emission threshold, to provide some control on the membrane-permeabilizing stress experienced by cells in a cavitation field is discussed.