A REVIEW AND APPLICATION OF CRYOPROTECTANT: THE SCIENCE OF CRYONICS

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ABOUT AUTHORS:
Ankit J. Joshi
Department of Pharmaceutics & Pharmaceutical Technology,
S. K. Patel College of Pharmaceutical Education and Research,
Ganpat University, Ganpat vidyanagar, Kherva, Mehsana-Gozaria Highway, Gujarat.
Joshiankit2824@gmail.com

ABSTRACT
The preservation of cells, tissues and organs by cryopreservation is promising technology now days and Low temperature technology has progressed in the field of tissue engineering, food preservation, fertility preservation, making disease resistant breeds since the early years to occupy a central role in this technology. Cryopreservation is important technology in every field like in organ cryopreservation, food cryopreservation, human cryopreservation, seeds cryopreservation, protein cryopreservation and pharmaceuticals. Cryobiologists will be required to collaborate with new physical and molecular sciences to meet this challenge. How cryoprotectants work is a mystery to most people. In fact, how they work was even a mystery to science until just a few decades ago. This article will explain in basic terms how cryoprotectants protect cells from damage caused by ice crystals, and with some of the advances.

REFERENCE ID: PHARMATUTOR-ART-2381

PharmaTutor (Print-ISSN: 2394 - 6679; e-ISSN: 2347 - 7881)

Volume 4, Issue 1

Received On: 12/08/2015; Accepted On: 27/08/2015; Published On: 01/01/2016

How to cite this article: Joshi AJ; A Review and Application of Cryoprotectant: The Science of Cryonics; PharmaTutor; 2016; 4(1); 12-18

INTRODUCTION
A cryoprotectant is a substance used to protect biological tissue from freezing damage (i.e. that due to ice formation). Cryopreservation is a process where cells or tissues are preserved by cooling to low sub-zero temperatures, such as 77 K or -196°C (the boiling point of liquid nitrogen). Cryopreservation is the use of very low temperatures to preserve structurally intact living cells and tissues. Unprotected freezing is normally lethal. Theories of freezing injury have envisaged either that ice crystals pierce or tease apart the cells, destroying them by direct mechanical action, or that damage is from secondary effects via changes in the composition of the liquid phase. Cryoprotectants, simply by increasing the total concentration of all solutes in the system, reduce the amount of ice formed at any given temperature; but to be biologically acceptable they must be able to penetrate into the cells and have low toxicity.At these low temperatures the biochemical reactions is effectively stopped that would otherwise leads to damage to tissue or cell death. However, when cryoprotectant solutions are not used, the cells being preserved are damaged due to freezing or thawing during the approach to low temperatures or warming to room temperature [1, 2]

Types of cryopreservation[1-4]
(1) Isochoric cryopreservation

Isochoric (constant volume) cryopreservation is referred for biological material at low temperature. In isochoric cryopreservation, the solution concentration is lower than the isobaric cryopreservation at all temperature. Isochoric cryopreservation is very simple; freezing is done in a constant volume chamber.

Since the reduction of metabolic rates is a strong function of temperature, so result of decrease metabolic activity  for every 10 degree Celsius decrease in temperature it is most desirable to preserve biological materials close to absolute zero. In it the system naturally adjusts at the minimal pressure for the particular cryopreservation temperature. Using a simple isochoric cryopreservation device, we confirm the theoretical thermodynamic predictions.

(2) Isobaric cryopreservation
Isobaric (constant pressure) process, occur at a pressure of 1 atm. which is constant atmospheric temperature and also implement on Earth and reduce chemical damage at high subzero or negative temperatures by adding compounds that depress the freezing point of the solution, can penetrate the cell membrane.

(3) Hyperbaric cryopreservation
It also reduce the ionic concentration at subzero Celsius temperatures. Like isochoric cryopreservation, it also leads to elevated pressures, thus elevated pressures, followed by rapid freezing lead to reduction of ice crystal and preservation of the biological material structure. Several tissues are preserved by using hyperbaric pressure at low subzero temperature e.g., kidney, liver, cornea, blood cells or cell preservation. Survival of cells is directly proportional to magnitude of the pressure. As increases the pressure survival of cells also increases e.g. Kidney (1000 atm.), cells (200 atm.), liver (70MPa) pressure limit are adjusted.

Damage or risk during cryopreservation
The damage of cells during cryopreservation mainly occurs during the freezing stage include: solution effects, extracellular ice formation, dehydration and intracellular ice formation. Many of these effects can be reduced by Cryoprotectants. (Show figure 1)

Solution effects
The damage caused by chemical and osmotic effects of concentrated solutes in the residual unfrozen water between ice crystals. This is so-called “solution effects” injury. During freezing, ice crystals form internally that excludes the solutes which remains in external liquid as externally concentration of solute is high that causes damage.

Extracellular ice formation
At slow cooling rate, water migrates out of cells and ice crystal forms extracellular. This will cause mechanical damage to the cell.

Dehydration
The migration of water causing extracellular ice formation which will cause cellular dehydration.


Figure 1: Graphical presentation of damage

(a) Living tissue contains much of water. (b) Water is component of a cellular solution in living tissue. (c) When tissue is cooled below freezing temperature, water molecules gather together and form growing ice crystals. (d) Growing ice expel other molecules from the ice lattice to form a harmful concentrated solution. (e) On a cellular scale, ice forms first outsidecells. (f) Growing ice causes cells to dehydrate and shrink. (g) Finally cells are left damaged and squashed between ice crystals. The damage is mostly mechanical.

How Cryoprotectants Protect Cells:
1. Adding cryoprotectants to cellular solution can prevent crystallization of water to ice.
2. Instead of freezing, molecules just move slower and slower as they are cooled.
3. Finally, at very low temperature, molecules become locked in place and an amorphous solid is formed. Water that becomes solid without freezing is said to be "vitrified".
4. There is no damage to cells during cooling because no ice is formed.          

Mechanism of cryoinjury[5-7]
Mazur’s two-factor hypothesis revealed two mechanisms of cell injury during freezing and thawing;

One occurring at cooling rates where the cell remains close to osmotic equilibrium and the other at rates in which there is super cooled water within the cell.

Graded Freezing technique is used to separate above two type of injury. The graded freezing technique require following steps:
1. Sample is slowly cooled
2. Two sample is removed at different temperature
3 .one samples is thawed
4. Second sample is dip in liquid nitrogen

Main methods to prevent risks [10]

1) Controlled rate and slow freezing
This method involves a brief pre-equilibration of cells in cryoprotectant solutions followed by slow, gradual, controlled cooling at rates optimized for the type of cells being cryopreserved. The whole process is carried out with the use of special programmable cell freezing equipment and requires 3-6 hours to complete. Cryoprotectants are used to protect the cells from damage due to intracellular ice crystal formation. The temperature of the cells is lowered to a super cooled state and ice crystal growth is initiated within the extra-cellular solution by a process called seeding.

As the size of the ice crystals increases, water in the solution is converted from liquid state to solid. This increases the concentration of solute in the extracellular medium which draws water out of the cell. As a result the cell dehydrates and this increased intracellular solute concentration further lowers the freezing point of the cell to approximately -350 C. The cell is almost devoid of any water at this point and therefore ice crystal formation is negligible when the cell ultimately freezes at this temperature. The rate at which water leaves the cell depends on the rate of cooling.

When the cells are cooled at rapid rate, water present inside the cell will not be able to move out fast enough leading to the formation intracellular ice crystals which are lethal to the cell. If the cells are cooled too slowly, then there will be severe volume shrinkage leading to high intracellular solute concentration, which have deleterious effects on the lipid-protein complexes of cell membranes. In addition the cells that are cooled slowly are potentially affected by chilling injury.


Figure 2: Influence of rate of cooling on intracellular ice formation.

Hence the rate of cooling and cryoprotectant concentration employed in the protocol should be optimized to avoid the intracellular ice crystallization and high solute concentration, the two main events involved in cellular injury during cryopreservation.

The success of slow cooling depends on achieving this optimal balance between the rate at which water can leave the cell and the rate at which it is cooled before it is converted into ice. Therefore, to achieve this balance, the time to complete slow cooling procedures for human oocytes and embryos needs a minimum of 90 min. Besides the longer time needed, the slow cooling protocol requires expensive programmable freezing equipment. Apart from these limitations slow cooling protocols are not satisfactory for various types of cells viz, pig embryos, in vitro derived bovine embryos, human MII oocytes and blastocysts which are sensitive to chilling injury.

2) Flash-freezing process (vitrification)
This method of cryopreservation was developed to overcome the shortcomings of slow freezing protocol. It is the solidification of a solution at low temperature, not by ice crystallization, but by extreme elevation in its viscosity using high cooling rates of 15,000 to 30,0000 C/min. The cooling of cells at this ultra high rate of freezing creates a glass like state without intracellular ice formation. Thus, the term vitrification, which means ‘turned into glass’ was first proposed by Luyet (1937). During vitrification the viscosity of the cytosol becomes greater and greater until the molecules become immobilized and it is no longer a liquid, but rather has the properties of a solid’. [11]

Vitrification involves exposure of the cell to high concentration of cryoprotectants for a brief period at room temperature followed by rapid cooling in liquid nitrogen. The cells are initially pre-equilibrated in a cryoprotectant solution of lower strength (usually 10 %) resulting in dehydration of the cell and its permeation with cryoprotectant. This is followed by a very short incubation (<30 seconds) in higher concentration of cryoprotectant solution (40%) followed by rapid plunging into liquid nitrogen. The high osmolarity of the cryoprotectants results in complete dehydration of the cell. Since the cells are almost devoid of any water by the time they are immersed in liquid nitrogen, the remaining intracellular water, if any, does not form ice crystals. During warming the entire process of vitrification of the cell is reversed. Cells are exposed in a step wise manner to hypotonic solutions of decreasing strengths of sucrose to remove the cryoprotectant and gradually rehydrate.

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