• Process by which any living cells, tissues,
organs or entire bodies are protected from
decay by storing them at extremely low
• 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 reach
low temperatures without causing additional
damage caused by the formation of ice
• Traditional cryopreservation has relied on
coating the material to be frozen with a
class of molecules
• New methods are constantly being
investigated due to the inherent toxicity of
• According to the Cryonics Institute, the
fundamental goal is "to give people a
second chance at life".
• 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
• 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” =
• 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
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
By Hina Ahmed and Myra Akhtar
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.
• 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
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
• 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
FURTHERMORE, COMPARED TO
• 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
• 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
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, for example in
hematologic malignancies or breast cancer. 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
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
A preferred option, species-dependent, is
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)
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
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
Mycelia are macerated (not for use with human
pathogenic fungi) and mixed to make a final
concentration of 10% glycerol or 5% DSMO.
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
All antibiotics should be washed from the culture
before long-term storage
Deep freezing method
Bacteria can be frozen using a solution of 15%
The process is simple and requires screw cap
microfuge tubes and sterile glycerol.
The glycerol is diluted to 30% so that it is easy
Equal amounts of 30% glycerol and culture
broth are mixed, dispensed into tubes and
Cryonics(full human body conservation)
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
Cryonics regarded with skepticism
Cryonics is regarded with skepticism within the mainstream
scientific community and is not part of normal medical
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
By Ayesha Rehman and Javaria Khan
Media preparation for Cell And Tissue Cryopreservation
Thawing Cryopreserved Cells
Handle the cells gently during harvesting since
damaged cells will not survive the additional
damage that occurs during the freezing and
Media for Cell and Tissue Cryopreservation
A typical media contains 90% serum + 10%
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
Glycerol and DMSO are the most commonly employed
Fetal bovine serum (FBS) is often used in mammalian
cryopreservation solutions, but it is not a cryoprotective
Salts, such as magnesium chloride, have been reported to
be cryoprotective agents.
Dextrans, glycols, starches, sugars, and polyvinylpyrrolidone
provide considerable cryoprotection in a variety of biologic
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
• 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
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
Step Down Freezing
The 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
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
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
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.
The 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
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 water
bath) provides the best recovery for most cell cultures
as it reduces or prevents the formation of damaging
ice crystals within cells during rehydration.
Since some cryoprotective agents may damage cells
upon prolonged exposure, remove the agents as
quickly and gently as possible by centrifugation
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
Using appropriate cryoprotective agents
Maintaining appropriate storage temperatures
Controlling the warming rate
• Cryopreservation helps in the preservation of biological materials.
• Cryopreservation is used to maintain the biosynthetic properties of
• Sperm, gametes, embryos, tissues, bone marrow, organ can be
• Helps to study the adapting nature of plants and animals under the
• 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
• 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
• The second who are delaying their childbearing
for personal reasons.
Preservation of Micro-biology cultures
• Bacteria and fungi can be kept short term
• However, cell division and metabolism is not
• 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
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
• 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
• Less often, OHSS happens during fertility treatments using
medications you take by mouth, such as clomiphene (Clomid,
• Anti-nausea medication, prescription painkillers or both
• Frequent physical exams and ultrasounds
• Daily weigh-ins and waist measurements to check for drastic
• Measurements of how much urine you produce each day
• Blood tests to monitor for dehydration, electrolyte imbalance and
• Adequate fluid intake
• Drainage of excess abdominal fluid using a needle inserted in your
• Support stockings, to help prevent blood clots
Obstacles and Recent
stories and Conclusion
By Syeda Zomia Naqvi Shah
3/5/2017 Free Template from www.brainybetty.com 67
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
3/5/2017 Free Template from www.brainybetty.com 68
• Ice formation can result in the needle shaped
crystals resulting in the damage to cell
• Unequal distribution or over distribution of
• Moreover, thermal gradients can induce
mechanical stress due to uneven expansion or
contraction in the biomaterial.
3/5/2017 Free Template from www.brainybetty.com 69
• 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
3/5/2017 Free Template from www.brainybetty.com 70
3/5/2017 Free Template from www.brainybetty.com 71
What you can do waking up after 100 years
Let me just show you.
3/5/2017 Free Template from www.brainybetty.com 72
3/5/2017 Free Template from www.brainybetty.com 73
She also had her
doubts but she said
that we need to have
our faith in
technology. She was
curious to see future
Free Template from www.brainybetty.com 74
• 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
Anna Elisabeth Johansson Bågenholm
3/5/2017 Free Template from www.brainybetty.com 75
“I preserve people to cheat death.”
3/5/2017 Free Template from www.brainybetty.com 76
• CEO of ALCOR life Extension Foundation
• Existing Location: California
• $200,000.00 Whole Body Cryopreservation
Who is Max More
3/5/2017 Free Template from www.brainybetty.com 77
• The Cryonics Institute (CI) is an American
• Location : Clinton Township, Macomb County,
• As of December 31, 2016, The Cryonics Institute
has 1,630 members in total (including 145
preserved bodies & 135 Assoc. Members).
• Prize $40,000
3/5/2017 Free Template from www.brainybetty.com 78
3/5/2017 Free Template from www.brainybetty.com 79
Cryopreservation is preserving your blood, organs,
tissues, or even your whole body.
• Fear of Death
• Love of life
• Hope for a cure for their disease
• Curiosity about their future
• Or even wanting to be immortal
3/5/2017 Free Template from www.brainybetty.com 80
BUT As a Muslim we believe
So its Now up to you weather its useful or not.