Cryopreservation And Its Applications Overview

Aug 09, 2023, 16:45 IST

If you are looking for Cryopreservation, you have come to the right place!

The topic “Cryopreservation” will be covered in this article. Cryopreservation is preserving live cells, tissues, and other biological samples by placing them in a deep freezer at shallow temperatures. The sample is typically stored at 196°C or lower. All of a cell's biological processes end at such low temperatures, and the cell dies. The cell membrane may rupture due to ice buildup inside the cells. This can be avoided by carefully controlling the freezing rate and selecting the media.

This article will discuss the factors involving Cryopreservation steps, Cryopreservation of embryos, Oocyte Cryopreservation, Cryopreservation of Sperm, Benefits, Applications, and Advantages of Cryopreservation, Main Methods To Prevent Damage, Cryopreservation of Animal & Plant Cells and Background of cryoprotectants.

Introduction

Cells, tissues, or organs are frozen during the cryopreservation process to preserve the material for a long time. Any cell metabolism that can harm the biological material in issue is successfully stopped at low temperatures (usually 80 °C (112 °F) or 196 °C (321) °F using liquid nitrogen).

Long-distance biological sample transportation, long-term sample storage, and the creation of a sample bank are all made possible through cryopreservation. Cryoprotective agents (CPAs) are molecules added to the freezing process to lessen the osmotic shock and physical stresses that cells experience. Trees, wood frogs, and tardigrades are a few examples of plants and animals in nature with exceptional cold tolerance that are used as inspiration for some cryoprotective compounds in research.

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Cryopreservation Steps

The following are all the steps in preserving the obtained biological samples:

• Harvesting or Selection of material: When choosing biological materials, it's vital to consider a few factors such as volume, density, pH, shape, and damage-freeness.
• Addition of cryo-protectant: As they lower the medium's freezing point and enable a slower cooling rate, cryoprotective chemicals such glycerol, FBS, salts, sugars, and glycols are added to the samples to lower the risk of crystallization.
• Freezing: In this cryopreservation method, several freezing techniques are used to shield cells from harm and cell death caused by their contact with warm cryoprotective chemicals.
• Storage in liquid nitrogen: Before being transferred to the storage vessels, the cryopreserved samples are kept in a freezer at a temperature of -80°C for a minimum of 5 to 24 hours.
• Thawing: The warming of biological samples to regulate cooling and prevent crystallization-related cell damage.

Cryopreservation of Embryos

Hormones is utilized to treat infertility and promote egg production. After that, the eggs are removed and fertilized in a lab. It is possible to produce more embryos and implant them in the woman's uterus. These embryos can be cryopreserved and utilized in the future. The woman can receive a second embryo transfer without paying for a further IVF session.

Oocyte Cryopreservation

The vitrification procedure causes the eggs to freeze quickly, which gives ice crystal formation less time to occur. With a high concentration of antifreeze chemicals, new cryoprotectants are applied.

An antifreeze-like cryoprotectant bath with a low concentration is first used to soak the oocyte. Some sucrose is added to the egg to help draw out some water. The egg is next moved to a high-concentration antifreeze cryoprotectant for a brief period before being transferred to liquid nitrogen immediately. When the egg is defrosted and utilized to transplant the lady with the egg.

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Cryopreservation of Sperm

A solution is added to the semen sample to preserve it from freezing and thawing. Afterward, the sample is transferred to polypropylene vials and maintained in liquid nitrogen to freeze.

The sperm may also be placed in cryobanks for short-term freezing and storage. Later, this sperm may be applied to specific infertility therapies.

Benefits of Cryopreservation

• Fertility procedures.
• Space and labor requirements are minimal.
• Protection against genetic contamination.
• Protects the genetic integrity of priceless stains.
• Protects the genetic material of threatened species.
• Samples of living things can be kept for a longer time.
• Shields the samples from microbial contamination and illness.
• By freezing gametes, embryos, and other genetic material, reduces genetic drift.

Applications of Cryopreservation

The long-term storage method of cryopreservation is primarily used to preserve and sustain the viability of biological samples for an extended period.

Numerous fields utilize this preservation technique extensively, including cryosurgery, molecular biology, ecology, food science, plant physiology, and multiple medical applications. Other uses for the cryopreservation method include

1. Seed Bank.
2. Gene Bank.
3. Blood transfusion.
4. In vitro fertilisation.
5. Organ transplantation.
6. Artificial insemination.
7. Storage of rare germplasm.
8. Freezing of cell cultures.
9. Conservation of endangered plant species.
10. Biodiversity conservation.

Main Methods To Prevent Damages

The two primary methods for avoiding cryopreservation damages are vitrification, a more recent flash-freezing approach, and a well-established mix of controlled rate and delayed freezing.

• Slow programmable freezing

Through steady rate and slow freezing, commonly referred to as slow programmed freezing (SPF), cells are cooled to about -196 °C over several hours.

The first human frozen embryo was delivered in 1984 thanks to slow programmable freezing, which was first created in the early 1970s. Since then, devices that employ programmable sequences or controlled rates to freeze biological samples have been used for human, animal, and cell biology. These devices "freeze down" a selection before it is frozen, or cryopreserved, in liquid nitrogen to preserve it for ultimate thawing better. These devices are employed by medical facilities, veterinary clinics, and research facilities worldwide to preserve general tissue, oocytes, skin, blood products, embryos, sperm, and stem cells.

• Vitrification

Vitrification is a flash-freezing (ultra-rapid cooling) procedure that lessens the risk of cryopreservation damage and ice crystal formation.

In the middle of the 1980s, scientists Greg Fahy and William F. Rall contributed to the introduction of vitrification to reproductive cryopreservation. Researchers assert that as of 2000, vitrification offers the advantages of cryopreservation without causing harm because of ice crystal formation. With the advent of tissue engineering, the situation has become more complicated because, to maintain high cell viability and functions, construct integrity, and biomaterial structure, both cells and biomaterials must be kept free of ice.

Lilia Kuleshova was the first researcher to describe the vitrification of tissue-engineered constructions. She was also the first to vitrify oocytes, which led to a live delivery in 1999.

Advantages of Cryopreservation

• All removed and/or fertilized cells can be preserved for use in the future thanks to cryopreservation, which increases the effectiveness of assisted reproductive techniques.
• By freezing embryos in between cycles, ovarian stimulation is not necessary every time, and implantation can be delayed without wasting recovered oocytes if the woman's ovaries are overstimulated.
• Couples who become pregnant after their first treatment cycle can donate their unused frozen embryos to science thanks to cryopreservation.
• Currently, it is typical to implant just one or two embryos, with any extras being cryopreserved for use in subsequent treatment cycles.
• Through cryopreservation, persons losing their fertility can store their reproductive cells and possibly conceive using assisted reproductive technology. Women who desire to put off having children or who have a history of early menopause in their families may use it.

Cryopreservation of Animal Cells

Animal cell line creation is expensive and labor-intensive.

Compared to fertilizer cell lines, the continuous cell line has several advantages, including

• They are eternally alive.
• They develop more quickly.
• They are easier to clone.

Cryopreservation of Plant Cells

The need for genetic resource storage grows as economically, and uncommon species gradually become extinct. The convent journal technique of storage is ineffective at stopping losses brought on by

• Pathogen and pest attack
• Weather-related illnesses
• Disorder that results from nature
• Economic and political factors

Cryopreservation FAQs

Q1. What are cryopreservation's drawbacks?

Ans. The possibility of ice crystals forming inside the cells and resulting in cell damage is one of the technique's main drawbacks. The application of inappropriate cryoprotectants also impacts cell viability. Cellular dehydration and extracellular ice production can result from water movement.

Q2. What is cryopreservation's alternate name?

Ans. The term for this procedure is cryo-preservation or cryo-conservation. To obtain low temperatures without causing additional damage from ice crystal formation during freezing.

Q3. What impact does cryopreservation have?

Ans. The significant damage to cell membranes caused by cryopreservation alters the mitochondria's and cells' functional and metabolic condition. According to certain data, cryopreserved sperm exhibit higher levels of DNA single-strand breaks and DNA condensation or fragmentation.

Q4. Which solvent works best for cryopreservation, and why?

Ans. An all-purpose solvent, DMSO can stabilize cell membranes under varying circumstances, inhibiting the development of intracellular ice crystals during freezing and heat release during phase transitions.

Q5. Who made cryopreservation possible?

Ans. Polge et al. inadvertently discovered in 1948 that chicken spermatozoa will survive after being frozen to 70°C using glycerol, leading to cryopreservation's beginning. It is a technique for keeping cells alive at shallow temperatures, such as liquid nitrogen.

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