Cryopreservation

GROUP 5

INTRODUCTION

• Process by which any living cells, tissues,

organs or entire bodies are protected from

decay by storing them at extremely low

temperatures.

• Typically -80 °C using solid carbon dioxide or -

196 °C using liquid nitrogen.

• At low enough temperatures, any enzymatic or

chemical activity which might cause damage to

the biological material is effectively stopped.

• Cryopreservation methods seek to reachlow temperatures without causing additionaldamage caused by the formation of iceduring freezing.

• Traditional cryopreservation has relied oncoating the material to be frozen with aclass of moleculestermed Cryoprotectants.

• New methods are constantly beinginvestigated due to the inherent toxicity ofmany cryoprotectants

• According to the Cryonics Institute, thefundamental goal is "to give people asecond chance at life".

History

• Early theoreticians of cryopreservation was James Lovelock (born 1919) known for Gaia theory

• He suggested that damage to red blood cells during freezing was due to osmotic stress.

• 1949 – Ernest John Christopher Polge, was a English biologist who solve the mystery of how to preserve living cells and tissues at very low temperatures.

• He accidentally discovered the cryoprotective properties of glycerol on fowl sperm.

• 1953 – Jerome K. Sherman was a doctoral

candidate at the University of Iowa. His research

led him to successfully freezing and thawing

human sperm.

• He founded the world’s first sperm bank.

• 1964 – The term cryobiology was invented. It

can be literally translated as :

“cryo” = cold, “bios” = life, and “logos” =

Science

• 1988 – Yves Menezo is a French biologist who

gave his name to the first commercial culture

media used in in-vitro fertilization.

• 1995 – Edouard Servy and the biologist Zishu

Liu were the first in the world to successfully

transplant a cryopreserved blastocyst

following intracytoplasmic sperm injection.

• Companies offer the option of cryopreservation

to revive patients and even cure or treat the

diseases that killed them in order to give them a

new chance at life.

• The Cryonics Institute believes it is allowing

people to "buy time until technology catches up

and is able to fully repair and restore the human

body."

Why do people Go for It

• Fear of Death

• Love of life

• Hope for a cure for their disease

• Curiosity about their future

• Or even wanting to be immortal

How much does it cost

• The process is expensive. Fees start at $28,000

and go up to $200,000, paid upon death by

either the patient or their insurance policy.

• Companies often also require membership

ahead of the procedure and may apply

surcharges for people outside the country.

Types Of Cryopreservation

Neuropreservation:

The theory is that only the information stored in

the brain is important, and that a body to contain

the revived brain could be cloned or regenerated

in the future.

The goal of Neuropreservation is to preserve the

whole brain, as well as the nerve

connections of our most complex senses such

as vision and hearing, without injury.

Cont……

• The brain is kept enclosed inside the cranium

to prevent injuries caused by surgical removal.

• Thus the head is surgically removed from the

body at the sixth cervical vertebra in the neck.

• Neuroseparation, the isolation of the brain

from the body, has led to the mistaken idea

that Neuropreservation preserves “heads,”

however the main target of preservation is the

brain

HOW WILL NEUROPRESERVED

PATIENTS BE RECOVERED?• Regeneration has existed in nature for hundreds of millions of

years. Our cells have the ability to regenerate new organs,

tissues, and limbs. This complex program for regrowing parts

of or whole human bodies is encoded and lies dormant in our

genes.

• Extending these regenerative capabilities will allow for new

bodies to regrow around the preserved brain from single cells.

The new regenerated body will essentially be a younger,

healthier clone of the original body.

• Programming a brain to regrow a new body may seem

incredible, but nature already does things that are even more

incredible.

FURTHERMORE, COMPARED TO

CRYONICS...• It is less expensive for it cost less to maintain just

the brain than the whole body.

• The quality of brain preservation is much better in neuropatients. Cryoprotectants are more equally distributed through out the hemispheres of the brain and optimized without the interference of the other organs of the body.

• Newer and improve cryonic technologies are often made available to neuropatients before whole body patients.

Embryo cryopreservation:

• Cryopreservation of embryos is the process of

preserving an embryo at sub-zero temperatures,

generally at an embryogenesis stage corresponding to

pre-implantation, that is, from fertilization to

the blastocyst stage

• Embryo freezing is a great way to preserve your

fertility. Although women are most fertile from their teens

until age 35, that time frame is not always ideal for a

woman to start a family. Freezing eggs allows women to

harvest their eggs when they are most viable and

healthy.

Ovarian Tissue Cryopreservation:• Ovarian tissue

cryopreservation is cryopreservation of tissue of the ovary of a female.

• Cryopreservation of ovarian tissue is of interest to women who want fertility preservation beyond the natural limit, or whose reproductive potential is threatened by cancer therapy,[1] for example in hematologic malignancies or breast cancer.[2] It can be performed on prepubertal girls at risk for premature ovarian failure, and this procedure is as feasible and safe as comparable operative procedures in children.

Preservation of microbial culture

Bacteria and fungi can be kept short-term

refrigerated

Cell division and metabolism is not completely

arrested and thus is not an optimal option for long-

term storage (years) or to preserve cultures

genetically or phenotypically, as cell divisions can

lead to mutations or sub-culturing can cause

phenotypic changes.

A preferred option, species-dependent, is

cryopreservation.

Preservation of fungal culture Fungi, notably zygomycetes, ascomycetes and higher

basidiomycetes, regardless of sporulation, are able to be stored in liquid nitrogen or deep-frozen.

Cryopreservation is a hallmark method for fungi that do not speculate but have delicate spores are pathogenic or are to be used for genetic stocks.

As with many other organisms, cry protectants like DMSO or glycerol (e.g. filamentous fungi 10% glycerol or yeast 20% glycerol) are used.

Differences between choosing cry protectants are species dependent, but generally for fungi penetrating cry protectants like DMSO, glycerol or polyethylene glycol are most effective (other non-penetrating ones include sugars manifold, sorbitol, dextran, etc.

Non-sporulation fungi or embedded mycelia

10% glycerol is added to the tube and agar

plugs of fresh culture are added and

immediately frozen in liquid-nitrogen vapor

(−170 °C).

Cultures are thawed at 37 °C and plated.

Spores or mycelia from agar plate

10% glycerol or 5% DMSO spore or mycelia

suspension are made and frozen

Liquid mycelia

Mycelia are macerated (not for use with human

pathogenic fungi) and mixed to make a final

concentration of 10% glycerol or 5% DSMO.

Preservation of bacterial culture

Fresh culture plates

Deep freezing method

Fresh culture plates

From a fresh culture plate, one single colony of

interest is chosen and liquid culture is made.

From the liquid culture, the medium is directly

mixed with equal amount of glycerol; the colony

should be checked for any defects like

mutations.

All antibiotics should be washed from the culture

before long-term storage

Fresh culture plate method

Deep freezing method

Bacteria can be frozen using a solution of 15%

glycerol.

The process is simple and requires screw cap

microfuge tubes and sterile glycerol.

The glycerol is diluted to 30% so that it is easy

to pipette.

Equal amounts of 30% glycerol and culture

broth are mixed, dispensed into tubes and

frozen

Deep freezing method

Cryonics(human body preservation)

Diagrammatic representation

Cryonics(full human body conservation)

Introduction Cryonics (from Greek 'kayos-'

meaning 'cold') is the low-temperature preservation (usually at −196°C) of people who cannot be sustained by contemporary medicine, with the hope that resuscitation and restoration to full health may be possible in the far future.

Cryopreservation of humans is not reversible with present technology

Cryonicists hope that medical advances will someday allow cryopreserved people to be revived.

Diagrammatic representation

Cryonics regarded with skepticism Cryonics is regarded with skepticism within the mainstream

scientific community and is not part of normal medical practice.

Cryonics depends on beliefs that death is a process rather than an event, that clinical death is a prognosis of death rather than a diagnosis of death, and that the cryonics patient has not experienced information-theoretic death.

Cryonics procedures can only begin after legal death, and cryonics "patients" are considered legally dead.

Cryonics procedures ideally begin within minutes of cardiac arrest and use cryoprotectants to prevent ice formation during cryopreservation.

Basic Protocol

Cell Harvesting

Media preparation for Cell And Tissue Cryopreservation

Temperature

Freezing

Thawing Cryopreserved Cells

Storage

Viability Assessment

Cell Harvesting

Handle the cells gently during harvesting since

damaged cells will not survive the additional

damage that occurs during the freezing and

thawing processes.

Media for Cell and Tissue Cryopreservation

A typical media contains 90% serum + 10%

cryoprotectant.

CPRS

Cryoprotective agents reduce the freezing point of

the medium and also allow slower cooling rate,

greatly reducing the risk of ice crystal formation,

which can damage cells and cause cell death during

freezing.

Cryoprotectants

Glycerol and DMSO are the most commonly employedcryoprotective agents.

Fetal bovine serum (FBS) is often used in mammaliancryopreservation solutions, but it is not a cryoprotectiveagent.

Salts, such as magnesium chloride, have been reported tobe cryoprotective agents.

Dextrans, glycols, starches, sugars, and polyvinylpyrrolidoneprovide considerable cryoprotection in a variety of biologicsystems.

Types of Cryoprotectants

• Intracellular cryoprotectants with low molecular

weights that permeate cells. Intracellular

cryoprotectants, such as glycerol and dimethyl sulfoxide

at concentrations from 0.5 to 3 molar, are effective in

minimizing cell damage in many slowly frozen biological

systems.

• Extracellular cryoprotectants with relatively high

molecular weights that do not penetrate cells.

Extracellular cryoprotective agents, such as

polyvinylpyrrolidone and hydroxyethyl starch, are more

effective at protecting biological systems cooled at rapid

rates.

Temperature

When adding the cryopreservation media to the

sample it is important that the solutions be cold

(∼4°C). Cell exposure to warm solutions

containing DMSO can result in substantial cell

damage and death.

Freezing Methods for Cryopreservation

1. Step Down Freezing

2. Blast Freezing

3. Direct Plunge Freezing

4. Slow Freezing using a programmable freezer

5. Vitrification

Step Down FreezingThe samples are placed in a refrigerator overnight

at 4ᵒC, and then transferred to a -70ᵒC (-94ᵒF)

freezer for a period of time, and moved to cryo

storage. However ,this freezing process is time

consuming, difficult to repeat and document, and

does not provide the controlled cooling rates and

ice nucleation associated with a true controlled

rate freezer.

Blast Freezing

Blast freezing is a method designed for speed

rather than maximum viability, and it’s used to

decrease a specific volume of material by a set

temperature in a fixed amount of time. Blast

freezing is commonly used for large amounts of

material, like blood bags or large volumes of

protein. It’s important to note that there are

purpose-built blast freezers designed for this

process.

Direct Plunge Freezing

In Liquid Nitrogen Submersion, or Plunge

Freezing, samples are loaded into a heat block,

and that block is submerged into LN2 and then

placed in storage. This method has been used

successfully for small numbers of low volume

straws and vials.

Slow Freezing using a

Programmable Freezer

The cell vessels/vials are placed in freezer which

cools using cold nitrogen vapors. The temperature

inside the cooling chamber can be accurately

controlled and the time course of the temperature

can be programmed. However, the time course of

temperature inside the straws may be different due

to the generation of heat of fusion.

VitrificationThe term “Vitrification” refers to any process

resulting in “glass formation”, the transformation

from a liquid to a solid in the absence of

crystallization so the cells that are properly slow

frozen become “vitrified”. Vitrification involves

using high concentrations of cryoprotectants to

prevent ice formation but this technique is under

developed.

Storage

Store cryopreserved cells at minus 80ᵒC in freezer

for at least 4 hours and up to 24 hours prior to

transfer to an archive storage such as a freezer

capable of continually maintaining temperature

below minus 130°C or a gaseous phase liquid

nitrogen storage vessel.

Thawing/Warming Cryopreserved Cells

Rapid thawing (60 to 90 seconds at 37°C in waterbath) provides the best recovery for most cell culturesas it reduces or prevents the formation of damagingice crystals within cells during rehydration.

Since some cryoprotective agents may damage cellsupon prolonged exposure, remove the agents asquickly and gently as possible by centrifugation

Viability Assessment

Accurate sample assessment is critical to

determining preservation success and downstream

utility of cell and tissue systems, for both research

and clinical use. Comparing samples to pre-freeze

levels 1 day after thawing with metabolic or

biochemical assays provide more accurate

determinations of cell viability.

Variables to Optimize

Controlling the cooling rate by using an

appropriate freezer

Using appropriate cryoprotective agents

Maintaining appropriate storage temperatures

Controlling the warming rate

ADVANTAGES AND

DISADVANTAGES

Advantages • Cryopreservation helps in the preservation of biological materials.

• Cryopreservation is used to maintain the biosynthetic properties ofplants

• Sperm, gametes, embryos, tissues, bone marrow, organ can bepreserved.

• Helps to study the adapting nature of plants and animals under the low temperature.

• Used to preserve the genetic materials of the plants which are on the verge of extinction

• Prevent in breeding

• Reduced risk of microbial contamination

• Reduced risk of cross contamination with other cell lines

• Reduced risk of genetic drift and morphological changes

• Embryo cryopreservation is used most often to store

good quality excess embryos resulting from an IVF

treatment cycle.

• Embryos can be stored for a patient who elects to have

her eggs fertilized with donor sperms. Pregnancies have

been reported from embryos stored for 16 years.

• Human sperm cryopreservation is widely used to store

donor and partner spermatozoa to preserve sperms

• It also ensure the recovery of a small number of

spermatozoa in several male factor infertility17 .

• It is commonly called sperm-banking is a procedure to

preserve sperm cells.

Cryopreservation of oocyte

• Human oocyte cryopreservation is a new

technology in which a woman’s eggs are

extracted, frozen or stored.

• Egg freezing benefits two groups of women.

• One those who are diagnosed with a medical

condition

• The second who are delaying their childbearing

for personal reasons.

Preservation of Micro-biology cultures

• Bacteria and fungi can be kept short term

refrigerated

• However, cell division and metabolism is not

completely arrested

• It is not an optimal option for long term storage

genetically or phenotypically as cell divisions

can led to mutations.

In Medical Science

• Low temperature have been used in medicine

• Used to prevent food spoilage

• Now- a- days it is used in fertility treatment the

transport of human organs and the long- term

storage of biological specimens

• To conserve plant biodiversity

In Animal Husbandry:-

• The introduction of cryopreservation technology

leads a major breakthrough in animal husbandry7

.Since the 1st successful cryopreservation of bull

semen has been used to propagate the rare and

endangered species using assisted reproduction

techniques. Every year, more than 25 millions cows

are artificially inseminated with frozen-thawed bull

semen8 and many bovine calves have been

produced using the transfer of cryopreseved

embryos into cow

Disadvantages

There are few disadvantages to storing eggs.

• During the cycle where the eggs are harvested, patients

undergo a traditional IVF protocol.

• There are known side effects with fertility medication including

the risk of ovarian hyper stimulation syndrome or OHSS.

• The lengthy process of slow-rate freezing and the subsequent

long-term storage of these valuable cells can often be costly,

consuming large amounts of energy to accurately maintain

such low temperatures.

Ovarian hyper stimulation syndrome

(OHSS)• Ovarian hyper stimulation syndrome (OHSS) affects women taking

injectable hormone medications to stimulate the development of

eggs in the ovaries.

• This may occur in women undergoing in vitro fertilization (IVF),

ovulation induction or intrauterine insemination.

• Too much hormone medication in system can lead to OHSS, in

which ovaries become swollen and painful.

• A small number of women may develop severe OHSS, which can

cause rapid weight gain, abdominal pain, vomiting and shortness of

breath.

• Less often, OHSS happens during fertility treatments using

medications you take by mouth, such as clomiphene (Clomid,

Serophene).

Treatment• Anti-nausea medication, prescription painkillers or both

• Frequent physical exams and ultrasounds

• Daily weigh-ins and waist measurements to check for drastic

changes

• Measurements of how much urine you produce each day

• Blood tests to monitor for dehydration, electrolyte imbalance and

other problems

• Adequate fluid intake

• Drainage of excess abdominal fluid using a needle inserted in your

abdominal cavity

• Support stockings, to help prevent blood clots

Obstacles and Recent

stories and Conclusion

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Obstacles In Cryopreservation

• Upto 60% human body is composed of water.

What’s the issue then?

• Actually the freezing point of water is 0 degree

centigrade while the cryoscopy temperature can

be as low as -90 degree centigrade.

• Very expensive Technique

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• Ice formation can result in the needle shaped

crystals resulting in the damage to cell

membrane.

• Unequal distribution or over distribution of

cryoprotectants.

• Moreover, thermal gradients can induce

mechanical stress due to uneven expansion or

contraction in the biomaterial.

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• The cooling rate required for optimal survival

varies by several orders of magnitude between

different cell types.

• Mass transfer limitations

A 14-YEAR-OLD GIRL WITH TERMINAL

CANCER WON THE RIGHT TO HAVE HER BODY

CRYOPRESERVED. SHE WROTE A LETTER TO

THE COURT , SAYING THAT

FUTURE OF

CRYOPRESERVATION

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What you can do waking up after 100 years

?

Let me just show you.

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JUST SLEEPPP for 1000 of years

And wake up in new World

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KIM SUAZY

She also had her

doubts but she said

that we need to have

our faith in

technology. She was

curious to see future

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• A Swedish radiologist from Vänersborg, who

survived after a skiing accident in 1999.

• She was left trapped under a layer of ice for

80 minutes in freezing water.

• After rescue, Bågenholm was transported to

the Tromsø University Hospital, where a team

of more than a hundred doctors and nurses

worked in shifts for nine hours to save her life.

• Bågenholm woke up ten days after the

accident,

Anna Elisabeth Johansson Bågenholm

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“I preserve people to cheat death.”

Max More

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• CEO of ALCOR life Extension Foundation

• Existing Location: California

• $200,000.00 Whole Body Cryopreservation

Who is Max More

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• The Cryonics Institute (CI) is an American

member-owned-and-operated not-for-profit

corporation

• Location : Clinton Township, Macomb County,

Michigan

• As of December 31, 2016, The Cryonics Institute

has 1,630 members in total (including 145

preserved bodies & 135 Assoc. Members).

• Prize $40,000

CRYONICS INSTITUTE

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Any Questions??????