Feasibility of high pressure freezing with freeze substitution after long-term storage in chemical fixatives

Background: Human milk banks have a pivotal role in provide optimal food for those infants who are not fully breastfeed, by allowing human milk from donors to be collected, processed and appropriately distributed. Donor human milk (DHM) is usually preserved by Holder pasteurization, considered to be the gold standard to ensure the microbiology safety and nutritional value of milk. However, as stated by the European Milk Banking Association (EMBA) there is a need to implement the improvement of the operating procedure of human milk banks including preserving and storing techniques. Aim: The purpose of this study was to assess the effectiveness and safety of the selected new combination of methods for preserving donor human milk in comparison with thermal treatment (Holder pasteurization). Methods: We assessed (1) the concentration of bioactive components (insulin, adiponectin, leptin, activity of pancreatic lipase, and hepatocyte growth factor) and (2) microbiological safety in raw and pasteurized, high-pressure processed and lyophilization human breast milk. Results: The combination of two techniques, high-pressure processing and freeze-drying, showed the best potential for preserving the nutritional value of human milk and were evaluated for microbiological safety. Microbiological safety assessment excluded the possibility of using freeze-drying alone for human milk sample preservation. However, it can be used as a method for long-term storage of milk samples, which have previously been preserved via other processes. Conclusion: The results show that high-pressure treatment is the best method for preservation that ensures microbiological safety and biological activity but subsequent freeze-drying allowed long-term storage without loss of properties.

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The Use of Ultrarapid Freezing and Freeze Substitution to Verify Vitrification and/or Ice Formation in Vascular Tissue

Microscopy Today ◽

10.1017/s1551929500059381 ◽

2000 ◽

Vol 8 (9) ◽

pp. 16-21

Author(s):

Fred Lightfoot ◽

Michael Taylor ◽

Kelvin G.M. Brockbank ◽

Cindy Hastings

Keyword(s):

Scientific Community ◽

Vascular Tissue ◽

Freeze Substitution ◽

Ice Formation ◽

Smooth Muscle Tissue ◽

Long Term Storage ◽

Ultrarapid Freezing ◽

Term Storage

The expanding field of cryobiology, in particular that of the study of vitrification for long term storage of tissues for transplantation, has demonstrated that ice is damaging to smooth muscle tissue. Consequently conventional methods such as ultrarapid freezing and freeze substitution are becoming routine protocols to determine the quality of cryopreservation. This article introduces the scientific community to the PS-1000 cryofixation unit (Delaware Diamond Knives, Wilmington, DE) which provides both ultrarapid freezing and a means of validation of freeze substitution methods. When any tissue is frozen or vitrif ed for clinical use it is imperative to know the structural and functional integrity of these tissues. Ice formation within the extracellular matrix and dehydration of multi-cellular tissues, using conventional cryopreservation, is the principal reason why these methods frequently prove to be ineffective.

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High-pressure freezing of cells in suspension for low-voltage scanning electron microscopy (LVSEM)

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084624 ◽

1991 ◽

Vol 49 ◽

pp. 64-65

Author(s):

Marek Malecki ◽

James Pawley ◽

Hans Ris

Keyword(s):

Electron Microscopy ◽

High Pressure ◽

Low Voltage ◽

Culture Media ◽

Cell Structure ◽

Freeze Substitution ◽

Ice Crystal ◽

High Pressure Freezing ◽

Cell Culture Media ◽

Voltage Scanning

The ultrastructure of cells suspended in physiological fluids or cell culture media can only be studied if the living processes are stopped while the cells remain in suspension. Attachment of living cells to carrier surfaces to facilitate further processing for electron microscopy produces a rapid reorganization of cell structure eradicating most traces of the structures present when the cells were in suspension. The structure of cells in suspension can be immobilized by either chemical fixation or, much faster, by rapid freezing (cryo-immobilization). The fixation speed is particularly important in studies of cell surface reorganization over time. High pressure freezing provides conditions where specimens up to 500μm thick can be frozen in milliseconds without ice crystal damage. This volume is sufficient for cells to remain in suspension until frozen. However, special procedures are needed to assure that the unattached cells are not lost during subsequent processing for LVSEM or HVEM using freeze-substitution or freeze drying. We recently developed such a procedure.

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High-pressure freezing and freeze substitution of plant tissue

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124148 ◽

1992 ◽

Vol 50 (1) ◽

pp. 748-749

Author(s):

William P. Sharp ◽

Robert W. Roberson

Keyword(s):

High Pressure ◽

Cell Structure ◽

Leaf Tissue ◽

Freeze Substitution ◽

Crystal Formation ◽

Ice Crystal ◽

High Pressure Freezing ◽

Cell Components ◽

Cold Metal ◽

Brome Grass

The aim of ultrastructural investigation is to analyze cell architecture and relate a functional role(s) to cell components. It is known that aqueous chemical fixation requires seconds to minutes to penetrate and stabilize cell structure which may result in structural artifacts. The use of ultralow temperatures to fix and prepare specimens, however, leads to a much improved preservation of the cell’s living state. A critical limitation of conventional cryofixation methods (i.e., propane-jet freezing, cold-metal slamming, plunge-freezing) is that only a 10 to 40 μm thick surface layer of cells can be frozen without distorting ice crystal formation. This problem can be allayed by freezing samples under about 2100 bar of hydrostatic pressure which suppresses the formation of ice nuclei and their rate of growth. Thus, 0.6 mm thick samples with a total volume of 1 mm3 can be frozen without ice crystal damage. The purpose of this study is to describe the cellular details and identify potential artifacts in root tissue of barley (Hordeum vulgari L.) and leaf tissue of brome grass (Bromus mollis L.) fixed and prepared by high-pressure freezing (HPF) and freeze substitution (FS) techniques.

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