Cryopreserved

  • What Is Cryopreserved ?
  • Cryopreserved Procedure
  • Why Choose Kasturi Cryopreserved ?
  • Do's And Dont's

Organelles, cells, tissues, and other biological constructions can all be preserved by a method called cryopreservation by being cooled to extremely low temperatures. The reactions of living organisms to ice formation are both theoretically fascinating and practically useful. Because ice crystal formation, osmotic shock, and membrane damage during freezing and thawing would cause cell death, stem cells and other viable tissues, which have great potential for use in basic research and for many medical applications, cannot be stored with simple cooling or freezing for a long time. With the aid of cryoprotective chemicals and temperature control apparatus, successful cryopreservation of cells and tissues has been steadily rising in recent years.Successful cryopreservation of cells or tissues and their clinical applications will require ongoing study of the physical and chemical features that happen during the freezing and thawing cycle. We briefly discuss representative cryopreservation techniques, like gradual freezing and vitrification, as well as the various cryoprotective chemicals in this overview. Cryopreservation's negative effects are also mentioned.

After people have therapies to try to get pregnant, embryo freezing frequently takes place. Examples include intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) (ICSI).

These techniques create more embryos by fertilizing eggs with sperm. Choosing to freeze excess embryos for future use is an option if:

  • Delay or cancel the implantation of a fertilized egg into your uterus.
  • Want to put off IVF till later.
  • Desire a backup plan in case initial fertility treatments don't work.
  • Rather than disposing of discarded embryos, consider donating them to researchers or other women who are trying to conceive.

The method of freezing and storing embryos for eventual use is known as embryo cryopreservation, sometimes known as embryo freezing. An egg that has been fertilised by a sperm is called an embryo. People can benefit from this procedure in terms of reproduction and fertility.

Another method of preserving fertility is embryo freezing. For instance, a cancer patient who is a woman or a person designated as female at birth (DFAB) may choose to rescue fertilised eggs before beginning chemotherapy or radiation therapy if such treatments may impair their capacity to conceive. Before beginning hormone treatments for the transition or getting gender-affirming surgery, a transgender guy (who is transitioning to male) may also preserve eggs or embryos.

The main goal of embryo freezing is to preserve the embryo for future use. As a result, the embryo must be kept in an environment that prevents its demise.

The following procedures are involved in freezing or cryopreserving the embryo:

Step one: Embryo Selection

The lady is treated medically to encourage her to produce more eggs as part of the IVF procedure. Additionally, the man produces more sperm cells. The development of several embryos is common when both are combined. One to four embryos are often transferred into the lady by the doctor, who discards the remainder. The healthiest embryo or embryos are chosen in this case so they can be saved for later use.

Step two: Removal of the water content

The chosen embryo cannot be immediately frozen without refilling the water in the cells. This is due to the fact that when it is frozen, the water content within the cells also freezes and crystalizes. The crystallisation process may result in enlargement, which may cause the cell to burst open and die. Cryoprotectants are employed to refill the water content in the cells in order to stop this from happening.

Step three: Embryo Freezing

The embryo is chilled to 20 degrees Celsius and then frozen as the water content is eliminated. One of these two approaches can be used to accomplish this:

  • Slow freezing

In this procedure, the embryos are placed within a tube made of straw that has been sealed with sterilised bearings. To avoid ageing and damage, the temperature in the tubes is gradually decreased (from 20° to -7° and finally to -35°) after the embryos are inside. The straw or tube is taken out of the freezer and kept at a temperature of -196 degrees Celsius in the liquid nitrogen container.

Kasturi provides a number of specialist investigative methods for male and female infertility, providing couples the best opportunity possible of becoming pregnant. Kasturi brings unmatched dedication to productive results to the table.

Combining a patient-centric approach with a team of subject-matter specialists has proven to be successful for us. Kasturi provides you with the knowledge and technology to improve your chances of becoming pregnant via the use of cutting-edge technologies and carefully supervised clinical practises.

Fertility, reproductive medicine, reproductive endocrinology, andrology, urology, fertility enhancing laparoscopic surgeons, foetal medicine, as well as a supportive group of physiotherapists, counsellors, and dieticians, make up our team.

For the first visit, during which a thorough history and prior reports are examined, both partners are anticipated to attend. In order to comprehend your medical history and formulate a treatment plan for you, a thorough medical history will be gathered along with a few blood tests. Simple solutions are prioritised, and your care plan will be developed based on the details of your unique circumstance, which will be disclosed to you.

Successful cryopreservation depends on a number of variables, including cell collection, cryoprotective agents (CPAs), storage vessels, cooling rate, cryogenic storage, and thawing. Unfortunately, during the thawing and plating processes, we frequently observe cell viability issues that are related to cryopreservation. I'd recommend focusing on these four crucial factors to increase cell viability.

  • Priority one: When cells are frozen, cell health and density are crucial. The better post-thawing vitality you may acquire, the healthier your cells are. In order to fill a normal Corning® cryogenic vial, we advise 2 x 106 cells (Corning Cat. No. 430661 or 430489). The nutrients or CPAs might not be enough to keep the health of the cells if the cell density is too high. Additionally, the cells must be kept away from excessive exposure to CPAs or cell dissociation chemicals before freezing, as well as prolonged room temperature storage when cell harvesting.
  • Second, the chilling rate (-1°C per minute) is crucial for transferring cryogenic vials containing resuspended cells with freezing media into a freezing container such a Corning CoolCellTM Container at -80°C. When freezing cells, you must use the right freezing container, CPA type, and concentration.
  • Third, you should keep the cells away from warm environments while transferring them to cryogenic storage (-196°C).

       •   Lastly, to prevent toxicity or osmotic shock, CPAs must be properly removed and thawed rapidly. I just gave a webinar presentation on how to store animal cell cultures using cryogenics. Please refer to the webinar that is                         accessible through the Corning website. Pay close attention to time mark 25:27 when I discuss a troubleshooting procedure that can increase your viability.

When thawing the iPSC:

  • Cryovials should be quickly defrosted by putting them in a water bath that is heated to 37°C.
  • Drop by drop, transfer the cell suspension using a pipettor into a 10X volume of media. The procedure ought to be delicate and gradual.
  • Each 35mm well (6-well plate) can have a seeding density of 2x105 to 1x106 viable cells. After 30 minutes of freezing, cells may adhere to the Corning Matrigel® coated plate. After 24-48 hours after plating, 70-80% confluence may be seen.
  • A healthy cell state. Before cryopreservation, iPSC should be fed regularly to ensure a healthy condition. Cells should be frozen two to four days after passage. After thawing, overgrowth can reduce viability. Cell clumps need to be broken up prior to cryopreservation.
  • In the long run, I would advise cryopreserving the iPCS in the vapour phase as opposed to liquid nitrogen. The vapour phase has a temperature range of -140°C to -180°C. Mammalian cells can also be kept at -80°C, although after a few months of storage, the vitality of the cells starts to decline. According to studies, compared to liquid nitrogen storage, adding 10% Ficoll 70 to 10% DMSO-containing cryoprotectant keeps cells frozen at -80°C for a year without losing viability. You can read the paper below for more information.

Dr. M. Kalpana (DNB, OBGYN, Obstetrician & Gynaecologist Fertility Specialist Fetal Medicine Specialist)

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