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A Review and Application of Cryoprotectant: The Science of

Ankit J. Joshi Department of Pharmaceutics & Pharmaceutical Technology, S. K. Patel College of Pharmaceutical Education and Research, Ganpat University, Kherva, Gujarat. [email protected]

ABSTRACT The preservation of cells, tissues and organs by is promising technology now days and Low temperature technology has progressed in the field of , 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 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 crystals, and with some of the advances.

Key Words: Cryopreservation, Cryoprotectants, , Penetrating & non-penetrating cryoprotectant.

INTRODUCTION A cryoprotectant is a substance used to protect Types of cryopreservation [1-4] biological tissue from damage (i.e. that due (1) Isochoric cryopreservation to ice formation). Cryopreservation is a process Isochoric (constant volume) cryopreservation is where cells or tissues are preserved by cooling to low referred for biological material at low temperature. sub-zero temperatures, such as 77 K or -196°C (the In isochoric cryopreservation, the solution boiling point of liquid ). Cryopreservation is concentration is lower than the isobaric the use of very low temperatures to preserve cryopreservation at all temperature. Isochoric structurally intact living cells and tissues. cryopreservation is very simple; freezing is done in a Unprotected freezing is normally lethal. Theories of constant volume chamber. freezing injury have envisaged either that ice crystals Since the reduction of metabolic rates is a strong pierce or tease apart the cells, destroying them by function of temperature, so result of decrease direct mechanical action, or that damage is from metabolic activity for every 10 degree Celsius secondary effects via changes in the composition of decrease in temperature it is most desirable to the liquid phase. Cryoprotectants, simply by preserve biological materials close to absolute zero. increasing the total concentration of all solutes in the In it the system naturally adjusts at the minimal system, reduce the amount of ice formed at any pressure for the particular cryopreservation given temperature; but to be biologically acceptable temperature. Using a simple isochoric they must be able to penetrate into the cells and cryopreservation device, we confirm the theoretical have low . At these low temperatures the thermodynamic predictions. biochemical reactions is effectively stopped that would otherwise leads to damage to tissue or cell (2) Isobaric cryopreservation death. However, when cryoprotectant solutions are Isobaric (constant pressure) process, occur at a not used, the cells being preserved are damaged due pressure of 1 atm. which is constant atmospheric to freezing or thawing during the approach to low temperature and also implement on Earth and temperatures or warming to room temperature [1, 2] reduce chemical damage at high subzero or negative How to cite this article: Joshi AJ; A Review and Application of Cryoprotectant: The Science of Cryonics; PharmaTutor; 2016; 4(1); 12-18 Vol. 4, Issue 1 | magazine.pharmatutor.org

PharmaTutor PRINT ISSN: 2394-6679 | E-ISSN: 2347-7881 13 temperatures by adding compounds that depress the effects, extracellular ice formation, dehydration and freezing point of the solution, can penetrate the cell intracellular ice formation. Many of these effects can membrane. be reduced by Cryoprotectants. (Show figure 1)

(3) Hyperbaric cryopreservation Solution effects It also reduce the ionic concentration at subzero The damage caused by chemical and osmotic effects Celsius temperatures. Like isochoric of concentrated solutes in the residual unfrozen cryopreservation, it also leads to elevated pressures, water between ice crystals. This is so-called “solution thus elevated pressures, followed by rapid freezing effects” injury. During freezing, ice crystals form lead to reduction of ice crystal and preservation of internally that excludes the solutes which remains in the biological material structure. Several tissues are external liquid as externally concentration of solute preserved by using hyperbaric pressure at low is high that causes damage. subzero temperature e.g., , , cornea, blood cells or cell preservation. Survival of cells is Extracellular ice formation directly proportional to magnitude of the pressure. At slow cooling rate, water migrates out of cells and As increases the pressure survival of cells also ice crystal forms extracellular. This will cause increases e.g. Kidney (1000 atm.), cells (200 atm.), mechanical damage to the cell. liver (70MPa) pressure limit are adjusted. Dehydration DAMAGE OR RISK DURING CRYOPRESERVATION The migration of water causing extracellular ice The damage of cells during cryopreservation mainly formation which will cause cellular dehydration. occurs during the freezing stage include: solution

(a) (b) (c) (d)

(e) (f) (g) 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 outside cells. (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.

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HOW CRYOPROTECTANTS PROTECT CELLS increased intracellular solute concentration further 1. Adding cryoprotectants to cellular solution can lowers the freezing point of the cell to approximately prevent crystallization of water to ice. -350 C. The cell is almost devoid of any water at this 2. Instead of freezing, molecules just move slower point and therefore ice crystal formation is negligible and slower as they are cooled. when the cell ultimately freezes at this temperature. 3. Finally, at very low temperature, molecules The rate at which water leaves the cell depends on become locked in place and an amorphous solid is the rate of cooling. formed. Water that becomes solid without freezing is said to be "vitrified". When the cells are cooled at rapid rate, water 4. There is no damage to cells during cooling because present inside the cell will not be able to move out no ice is formed. fast enough leading to the formation intracellular ice crystals which are lethal to the cell. If the cells are MECHANISM OF CRYOINJURY [5-7] cooled too slowly, then there will be severe volume Mazur’s two-factor hypothesis revealed two shrinkage leading to high intracellular solute mechanisms of cell injury during freezing and concentration, which have deleterious effects on the thawing; lipid-protein complexes of cell membranes. In One occurring at cooling rates where the cell addition the cells that are cooled slowly are remains close to osmotic equilibrium and the other potentially affected by chilling injury. 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 Figure 2: Influence of rate of cooling on intracellular ice formation. [10] MAIN METHODS TO PREVENT RISKS 1) Controlled rate and slow freezing Hence the rate of cooling and cryoprotectant This method involves a brief pre-equilibration of cells concentration employed in the protocol should be in cryoprotectant solutions followed by slow, optimized to avoid the intracellular ice crystallization gradual, controlled cooling at rates optimized for the and high solute concentration, the two main events type of cells being cryopreserved. The whole process involved in cellular injury during cryopreservation. is carried out with the use of special programmable The success of slow cooling depends on achieving cell freezing equipment and requires 3-6 hours to this optimal balance between the rate at which complete. Cryoprotectants are used to protect the water can leave the cell and the rate at which it is cells from damage due to intracellular ice crystal cooled before it is converted into ice. Therefore, to formation. The temperature of the cells is lowered to achieve this balance, the time to complete slow a super cooled state and ice crystal growth is cooling procedures for human oocytes and embryos initiated within the extra-cellular solution by a needs a minimum of 90 min. Besides the longer time process called seeding. needed, the slow cooling protocol requires expensive programmable freezing equipment. Apart As the size of the ice crystals increases, water in the from these limitations slow cooling protocols are not solution is converted from liquid state to solid. This satisfactory for various types of cells viz, pig increases the concentration of solute in the embryos, in vitro derived bovine embryos, human extracellular medium which draws water out of the MII oocytes and blastocysts which are sensitive to cell. As a result the cell dehydrates and this chilling injury.

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2) Flash-freezing process (vitrification) most commonly used cryoprotectants are classes of This method of cryopreservation was developed to cryoprotectants called penetrating cryoprotectants. overcome the shortcomings of slow freezing Penetrating cryoprotectants are small molecules that protocol. It is the solidification of a solution at low easily penetrate cell membranes. The molecular temperature, not by ice crystallization, but by mass of penetrating cryoprotectants is typically less extreme elevation in its viscosity using high cooling than 100 daltons. By entering and remaining inside rates of 15,000 to 30,0000 C/min. The cooling of cells cells, penetrating cryoprotectants prevent excessive at this ultra high rate of freezing creates a glass like dehydration of cells during the freezing process. state without intracellular ice formation. Thus, the term vitrification, which means ‘turned into glass’ COMPONENT OF CRYOPRESERVATION SOLUTION was first proposed by Luyet (1937). During [12-13] vitrification the viscosity of the cytosol becomes A cryopreservation solution, which may be either a greater and greater until the molecules become freezing solution or vitrification solution, consists of: immobilized and it is no longer a liquid, but rather has the properties of a solid’. [11] Carrier Solution: Carrier solution consists of solution ingredients that are not explicit cryoprotectants. The Vitrification involves exposure of the cell to high role of the carrier solution is to provide basic support concentration of cryoprotectants for a brief period at for cells at temperatures near freezing. It contains room temperature followed by rapid cooling in liquid salts, osmotic agents, pH buffers, and sometimes nitrogen. The cells are initially pre-equilibrated in a nutritive ingredients or apoptosis inhibitors. The cryoprotectant solution of lower strength (usually 10 ingredients are usually present at near isotonic %) resulting in dehydration of the cell and its concentration (300 milliosmoles) so that cells neither permeation with cryoprotectant. This is followed by shrink nor swell when held in carrier solution. Carrier a very short incubation (<30 seconds) in higher solution is sometimes called “base perfusate.” concentration of cryoprotectant solution (40%) The carrier solution typically used with M22 followed by rapid plunging into liquid nitrogen. The cryoprotectant solution is called LM5. Different high osmolarity of the cryoprotectants results in concentrations of cryoprotectant may be required at complete dehydration of the cell. Since the cells are various stages of cryoprotectant introduction and almost devoid of any water by the time they are removal, but the concentration of carrier solution immersed in liquid nitrogen, the remaining ingredients always remains constant. This constant- intracellular water, if any, does not form ice crystals. composition requirement can be regarded as the During warming the entire process of vitrification of definition of a carrier solution. As a practical matter, the cell is reversed. Cells are exposed in a step wise this means that cryopreservation solutions must be manner to hypotonic solutions of decreasing made by means other than adding cryoprotectants strengths of sucrose to remove the cryoprotectant to a pre-made carrier solution because native and gradually rehydrate. addition would dilute the carrier ingredients.

Properties of Cryoprotectants: Ice Blockers (Optional Ingredient): Ice blockers are Not all chemicals that dissolve in water are compounds that directly block ice growth by cryoprotectants. In addition to being water soluble, selective binding with ice or binding to contaminants good cryoprotectants are effective at depressing the that trigger ice formation (ice nucleators). melting point of water, do not precipitate or form Conventional cryoprotectants act by interacting with eutectics or hydrates, and are relatively non-toxic to water. Ice blockers compliment conventional cells at high concentration. cryoprotectants by interacting with ice or surfaces that resemble ice. Ice blockers are like drugs in that All cryoprotectants form hydrogen bonds with water. only a small amount is required to find and bind their Since the discovery of as the first target. Low molecular weight polyvinyl and cryoprotectant more than 50 years ago the best and polyglycerol, called X-1000 and Z-1000, and biological proteins are examples of ice

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PharmaTutor PRINT ISSN: 2394-6679 | E-ISSN: 2347-7881 16 blockers. Ice blockers are only used in vitrification APPLICATION & RECENT ADVANCES [14-18] solutions, not freezing solutions 1. Advances in organ freezing The main new initiatives in organ freezing have been CLASSIFICATION efforts to successfully freeze whole ovaries to These are usually separated into two broad classes preserve fertility against the risks posed by based on their ability to diffuse across cell chemotherapy in youth or by aging. Seven rat membranes. Penetrating cryoprotectant are able to ovaries, still attached to their fallopian tubes and the move across cell membranes whereas non- upper segment of the uterus, were perfused with penetrating agents cannot. 0.1M fructose and (DMSO) to attain a final DMSO concentration of 1.5M (just a bit Penetrating Cryoprotectant: Penetrating over 10% by volume) over 30 minutes, frozen slowly, cryoprotectants are small molecules able to cross and stored in liquid nitrogen overnight before cell membranes. The role of penetrating thawing, gradual cryoprotectant washout, and cryoprotectants is to reduce ice growth and reduce vascular transplantation. Three of seven thawed cell dehydration during freezing. In vitrification, the grafts had no follicles, and the 4 grafts that did have role of penetrating cryoprotectants is to completely follicles had reduced numbers of follicles and prevent ice formation. Penetrating cryoprotectants recipients had reduced estrogen levels, elevated FSH are the majority ingredients of vitrification solutions. levels, and reduced uterine weights, but the 4 (Ex.: DMSO) surviving ovaries were able to ovulate and one Non-Penetrating Cryoprotectants: Non-penetrating animal was able to get pregnant and have two pups cryoprotectants are large molecules, usually (reduced litter size). polymers, added to cryoprotectant solutions. They This was good enough for Nature, and, interestingly, inhibit ice growth by the same mechanisms as the microscopic structure of the fallopian tubes and penetrating cryoprotectants, but do not enter cells. uterine portion of the transplants was normal. Polyethylene glycol (PEG) and polyvinylpyrrolidone Clearly, freezing was detrimental, and the authors (PVP) are examples. Non-penetrating suggested that vitrification might work better, but cryoprotectants are usually less toxic than just as clearly, at least portions of these ovaries and penetrating cryoprotectants at the same all of the associated structures survived freezing and concentration. They reduce the amount of thawing. penetrating cryoprotectants needed by mimicking 2. Depressed Metabolism outside the cell the cryoprotective effects of proteins Cryopreservation may find the increasing use of the inside the cell. It has also been recently discovered term suspended animation for therapeutic that using non-penetrating cryoprotectants to interventions like whole body (ultra) profound increase the tonicity (osmotically active asanguineous hypothermia and normothermic concentration) of vitrification solutions can prevent a hypometabolism indicative of a lack of precision. But type of injury called chilling injury. (Ex.: Starch, the increased support for and research in these areas ) in mainstream biomedical science and the media may produce a more favorable reception of research Other examples based on carbohydrates and polyols: aimed at reversible human cryopreservation and real Cryoprotectant Examples suspended animation as a result. Another advantage Monosaccharide Fructose, Dextrose of increased research efforts in these areas is that Disaccharide Sucrose, lactose, inuline cryonics providers can benefit from these findings to Oligosaccharide Cellobiose,Trehalose enhance their own capabilities and initiate informed Polysaccharide Starch,Chitosan research into improved organ preservation solutions Amino acid Phenylalanin,Tyrosin and “hibernation mimetics.” Polyols Glycerol 3. Nosy Neuroprotection: Intranasal Delivery of Other Dimethyl sulfoxide (DMSO) and Neuroprotective Agents to the Brain its derivative In cryonics, certain neuroprotective agents are administered to patients in an attempt to prevent (or

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PharmaTutor PRINT ISSN: 2394-6679 | E-ISSN: 2347-7881 17 at least slow down) ischemic damage to the brain using traditional controlled-rate freezing and after cardiac arrest and during the low flow chemical additive cryoprotection. Pregrowth, reperfusion provided by cardiopulmonary support. pretreatment, and cold acclimation approaches for Cooling is, of course, our primary line of defense the improvement of tolerance to liquid nitrogen are against such damage because of its striking also presented. The article concludes by reporting an effectiveness in reducing metabolic demand. analytical protocol that profiles volatile hydrocarbon However, rapid field cooling still presents stress markers (for ethylene, hydroxyl radicals, and considerable logistical and clinical challenges and lipid peroxidation products) during cryopreservation. preferential brain cooling is (at least until the patient This method uses noninvasive headspace sampling arrives in the operating room) yet to be and gas chromatography, and it is widely applicable accomplished. across cryogenic systems. Therefore, quick and direct protection of the brain is 6. Cryopreservation of Red Blood Cells and Platelets especially important in the moments following Blood cells can be regarded as a classical field of pronouncement and during the initial stages of application of low-temperature . cooling, while the patient is still relatively warm. Cryopreservation methods have been developed for Currently, all medications delivered to cryonics different categories of blood cells namely red blood patients are introduced to the systemic circulation cells (RBCs) (erythrocytes), platelets (thrombocytes), either via the intravenous (IV) or intraosseous (IO) mononuclear cells (i.e., lymphocytes, monocytes), routes, requiring skilled personnel who have been and hematopoietic progenitor cells. trained in IV and/or IO technique. Due to the blood- 7. Cryopreservation of Mammalian Oocytes brain barrier and first-pass metabolism, relatively Two methods for the cryopreservation of large volumes of neuroprotective agents must be mammalian oocytes are described. One method uses given in order for an effective dose to enter the brain a relatively low concentration of the cryoprotectant via the systemic circulation. In fact, some of the most propanediol plus sucrose and requires controlled- promising neuroprotective drugs are currently not rate cooling equipment to achieve a slow cooling available at all for treatment of cryonics patients rate. Such a method has produced live births from because of poor BBB permeability. cryopreserved human oocytes. 4. Cryopreservation of Human Embryonic Stem Cell The second method described employs a high Lines concentration of the cryoprotectant dimethyl Two different approaches have been adopted for the sulfoxide plus a low concentration of polyethylene cryopreservation of human embryonic stem cells glycol. This method involves cooling by plunging (hESCs): vitrification and conventional slow standard straws into liquid nitrogen vapor, hence cooling/rapid warming. The vitrification method avoiding the need for specialized equipment,but described here is designed for hESCs that grow as requires technical ability to manipulate the oocytes discrete colonies on a feeder cell monolayer, and are quickly in the highly concentrated cryoprotectant subcultured by manual subdivision of the colonies solutions. Murine oocytes vitrified, using this into multicellular clumps. hESCs that are subcultured technique, have resulted in live births. by enzymatic dissociation can more conveniently be cryopreserved by conventional slow cooling/rapid CONCLUSION warming methods. Applied low temperature technology has progressed Although both methods are suitable for use in a significantly since the early years to occupy a central research context, neither is suitable for role in many modern scientific endeavors. While it cryopreservation of embryonic stem cells destined much has been learnt about the role of CPA and their for clinical diagnostic or therapeutic uses without mode of action, there still remain significant gaps in modification. our understanding about their molecular interactions 5. Cryopreservation of Shoot Tips and Meristems with cell components and potential . Shoot-tip meristem cryopreservation methodologies and cryonics are at the interface are reported for the complementary cryoprotective between physics, chemistry, and biology the strategies of vitrification and equilibrium freezing knowledge of physical-chemistry laws is very

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PharmaTutor PRINT ISSN: 2394-6679 | E-ISSN: 2347-7881 18 important. While at present there is much possibly be used to preserve smaller entities without uncertainty about whether cryonics is justifiable; a any damages. Being able to maintain vital organs personal choice is still the governing force in the (such as liver, lungs, heart, kidneys, skin, and bone matter. Cryobiologists will be required to collaborate marrow) in good conditions for extended periods with new physical and molecular sciences to meet would be remarkable advances in medicine, allowing this challenge. However, if improvements are made the creation of organ banks, and therefore reducing to the current cryopreservation techniques, it will the waiting time for .

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