INTRODUCTIONewingdigital.com/text_content/115985350295f47b575d4dd7.pdfEmbryo cryopreservation: •...
Transcript of INTRODUCTIONewingdigital.com/text_content/115985350295f47b575d4dd7.pdfEmbryo cryopreservation: •...
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??????