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Biomedical subjects

D R MacFarlane

Publications and source records attributed to D R MacFarlane.

8 recordsLinked to original sources
PubMed2005

Coral larvae conservation: physiology and reproduction.

Coral species throughout the world's oceans are facing severe environmental pressures. We are interested in conserving coral larvae by means of cryopreservation, but little is known about their cellular physiology or cryobiology. These experiments examined cryoprotectant toxicity, dry weight, water and cryoprotectant permeability using cold and radiolabeled glycerol, spontaneous ice nucleation temperatures, chilling sensitivity, and settlement of coral larvae. Our two test species of coral larvae, Pocillopora damicornis (lace coral), and Fungia scutaria (mushroom coral) demonstrated a wide tolerance to cryoprotectants. Computer-aided morphometry determined that F. scutaria larvae were smaller than P. damicornis larvae. The average dry weight for P. damicornis was 24.5%, while that for F. scutaria was 17%, yielding osmotically inactive volumes (V(b)) of 0.22 and 0.15, respectively. The larvae from both species demonstrated radiolabeled glycerol uptake over time, suggesting they were permeable to the glycerol. Parameter fitting of the F. scutaria larvae data yielded a water permeability 2 microm/min/atm and a cryoprotectant permeability = 2.3 x 10(-4) cm/min while modeling indicated that glycerol reached 90% of final concentration in the larvae within 25 min. The spontaneous ice nucleation temperature for F. scutaria larvae in filtered seawater was -37.8+/-1.4 degrees C. However, when F. scutaria larvae were chilled from room temperature to -11 degrees C at various rates, they exhibited 100% mortality. When instantly cooled from room temperature to test temperatures, they showed damage below 10 degrees C. These data suggest that they are sensitive to both the rate of chilling and the absolute temperature, and indicate that vitrification may be the only means to successfully cryopreserve these organisms. Without prior cryopreservation, both species of coral settled under laboratory conditions.

Animals↗
PubMed2004

Living cationic polymerisation of styrene in an ionic liquid.

For the first time, living cationic polymerisation of styrene has been carried out in room temperature ionic liquids under mild reaction conditions and using mild acid catalysts (e.g. organoborate acids) to obtain polymers of narrow polydispersity.

Journal Article↗
PubMed2002

Reversible self-polymerizing high T(g) lyoprotectants.

One mode of action of protectants in the storage of biological materials is by promoting the formation of a vitrified state on cooling or drying. In the case of preservation by drying, the glassy material comprises a low water content mixture of protectant and organic material. The protectant must on drying form a glassy state of glass transition temperature (T(g)) above the desired storage temperature. However, in some applications it must also be easily transported through cell membranes and this restricts the choice to a relatively limited number of small molecules, which typically exhibit very low glass transition temperatures. In this work we describe a self-polymerizing protectant comprising an inorganic salt and a small hydroxy functional molecule such as glycerol. This forms co-ordinate polymer chains of high T(g) on drying but rapidly depolymerizes into the original components on rehydration. The polymerization process is general for polyhydroxy compounds including glucose and related compounds.

Calcium Chloride↗
PubMed1999

Sugars exert a major influence on the vitrification properties of ethylene glycol-based solutions and have low toxicity to embryos and oocytes.

A systematic approach was taken to assess the vitrification properties of ethylene glycol-based solutions supplemented with carbohydrates. Solutions were prepared by weight (gravimetrically) using ethylene glycol as the cryoprotectant, 0.9% NaCl in water, and six different sugars: d-glucose, d(-)-fructose, d-sorbitol, sucrose, d(+)-trehalose, and raffinose. Sugars were added on a molal basis (0. 1, 0.5, and 1 m). Characteristics of the solutions were measured during warming by differential scanning calorimetry using a cooling rate of 100 degrees C/min and a warming rate of 10 degrees C/min. In the absence of carbohydrates a 59 wt% EG-saline solution formed a stable glass. When EG was replaced by an equimolal concentration of glucose, fructose, or sorbitol (monosaccharides) at 0.1, 0.5, or 1.0 m there was no change in the total solute concentration at which vitrification occurred, but the glass transition (Tg) occurred at a higher temperature than in EG-saline alone. When EG was replaced by an equimolal concentration of sucrose or trehalose (disaccharides) both the Tg and the lowest total solute concentration required for vitrification became progressively higher as the molecular weight, or the ratio of sugar to EG in the solutions, increased. At the highest tested disaccharide concentration (1 m) vitrification was achieved at a total solute concentration of 65 wt% (sucrose) and 67 wt% (trehalose). The polysaccharide raffinose significantly modified the vitrification properties of ethylene glycol solutions. When 0.5 or 0.1 m raffinose replaced EG on an equimolal basis the glass transition point was raised more than with either the monosaccharides or the disaccharides. Raffinose allowed vitrification at a total solute concentration of 67 wt% (0.5 m) and 63 wt% (0.1 m). The maturation of immature mouse oocytes, and the development of embryos in media containing 5-7 mM of any sugar was comparable to controls, indicating that they are not toxic. Exposure of freshly collected GV or MII oocytes to sugar concentrations between 0.5 and 1.0 M, for up to 10 min had no significant effect on the proportion which subsequently formed two cells. We conclude that added sugars do contribute to a solutions overall vitrification properties, and their properties should be taken into consideration when vitrification solutions are being designed or modified.

Animals↗
PubMed1998

Crystallization of ice in aqueous solutions of glycerol and dimethyl sulfoxide 2: ice crystal growth kinetics.

The crystallization of ice in aqueous solutions of glycerol and dimethyl sulfoxide (Me2SO) has been studied using a combined DSC-video microscope technique. The solutions investigated were 50w/w% glycerol and 45w/w% Me2SO; both of these solutions have a solute concentration of approximately 16 mol%. The rates of growth of the external surfaces of ice crystals from both of these solutions were determined over broad temperature ranges. The growth rates were found to be generally independent of time, particularly at lower temperatures. The ice crystal growth rate in the glycerol solution became negligible at a significantly higher temperature than in the Me2SO solution. Addition of anti-freeze protein from the winter flounder at concentrations of 1.7 and 9.9 mg g-1 was found to have no significant effect on the ice crystal growth rates in 50w/w% glycerol solutions.

Animals↗
PubMed1997

Vitrification properties of solutions of ethylene glycol in saline containing PVP, Ficoll, or dextran.

Vitrification solutions which are used for cells or embryos generally contain cryoprotectant, physiological saline, and one or more macromolecular solutes. The macromolecules modify the vitrification tendencies of these solutions, but there is little detailed information on the vitrification properties of ethylene glycol solutions containing the additives PVP, Ficoll, and dextran. This study therefore added ethylene glycol to 0.9% NaCl in water (saline) and used differential scanning calorimetry to determine the lowest concentration at which the solution would remain vitreous when a warming rate of 10 degrees C/min was used. In the absence of other additives 59 wt% ethylene glycol (EG) in saline formed a stable glass. When ethylene glycol was replaced by the polymers Ficoll and/or dextran on a weight for weight basis, the resulting solution vitrified less readily than an EG-saline solution even though the total solute concentration was kept constant. The total solute concentration required to form a stable vitreous solution increased as the Ficoll 70,000 and 400,000 MW or dextran 78,000 MW content increased (5, 10, and 20 wt%). Ficoll and dextran had little or no effect on the glass transition and melting points of the solutions. In the presence of PVP vitrification occurred at a total solute concentration of 59 wt% (PVP 360,000 MW) or 60 wt% (PVP 40,000 MW) for all three tested PVP concentrations (5, 10, and 20 wt%). Although this indicates that PVP and EG have comparable vitrification properties, the melting and the glass transition temperature of the solutions rose as the PVP content increased. When 1 m sucrose was added to saline and 0, 5, 10, or 20 wt% PVP 40,000 MW vitrification was achieved with 31, 26, 23, and 15% EG, respectively, indicating that the total solute concentration required for vitrification could be estimated with reasonable accuracy from the sum of the individual components. We conclude that the tested polymers differ in how they interact with ethylene glycol-based vitrification solutions.

Calorimetry, Differential Scanning↗
PubMed1991

An association between chromosomal abnormalities in rapidly frozen 2-cell mouse embryos and the ice-forming properties of the cryoprotective solution.

This paper investigates the effect of straw handling on the viability of 2-cell mouse embryos rapidly frozen in dimethyl sulphoxide (DMSO) solutions. During the brief (3 min) equilibration step, straws were either rotated periodically to keep the embryos in suspension, or kept still to allow the embryos to settle onto the the inner surface of the straw. The effects of these straw movements were tested with cryoprotectant solutions containing 1.5, 3.0 or 4.5 M-DMSO. Rapidly cooled straws containing 4.5 M-DMSO vitrify throughout on cooling, but ice forms on warming. The survival and normality of embryos frozen in 4.5 M-DMSO was not influenced by straw handling as 91-92% formed blastocysts in vitro, 77-78% formed normal fetuses, and no chromosomal rearrangements were observed. In solutions containing less than 4.5 M-DMSO ice formation occurred throughout (1.5 M-DMSO), or in parts (3.0 M-DMSO) of the cryoprotectant during cooling. The viability of embryos frozen in 3.0 or 1.5 M-DMSO solutions was reduced both in vitro and in vivo and structural chromosome aberrations, predominantly tri- and quadri-radial rearrangements, were observed. The reduction in embryo viability, and the chromosomal damage was particularly pronounced in embryos frozen in 3.0 M-DMSO in straws which were rotated during the equilibration step (47% blastocysts, 15% fetuses, 77% chromosome rearrangements). The results indicate that rapid freezing of 2-cell mouse embryos in 4.5 M-DMSO is safe and efficient, whereas freezing at lower DMSO concentrations is associated with severe chromosome damage, and reduced viability in vitro and in vivo.

Animals↗