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    Cryopreservation: Anuja Kamath, M.SC Bangalore Assisted Conception Centre

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    Al Mamun
    Cryopreservation is the process of preserving living cells and tissues at low temperatures. It allows for cells, embryos, and other living matter to be stored viably for future use. There are two main methods - slow freezing and vitrification. Slow freezing involves cooling at controlled rates while crystals form, while vitrification avoids ice crystal formation for small volumes by extremely fast cooling rates. Key aspects that determine cell survival include the cryoprotectants used, cooling and warming rates, and minimizing osmotic stress. Cryopreservation is important in reproductive medicine for gamete and embryo banking and has applications in cancer treatment.

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    Cryopreservation: Anuja Kamath, M.SC Bangalore Assisted Conception Centre

    Uploaded by

    Al Mamun
    68 views50 pages
    Cryopreservation is the process of preserving living cells and tissues at low temperatures. It allows for cells, embryos, and other living matter to be stored viably for future use. There are two main methods - slow freezing and vitrification. Slow freezing involves cooling at controlled rates while crystals form, while vitrification avoids ice crystal formation for small volumes by extremely fast cooling rates. Key aspects that determine cell survival include the cryoprotectants used, cooling and warming rates, and minimizing osmotic stress. Cryopreservation is important in reproductive medicine for gamete and embryo banking and has applications in cancer treatment.

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    Techniques of Cryopreservation

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    Cryopreservation

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    Cryopreservation is the process of preserving living cells and tissues at low temperatures. It allows for cells, embryos, and other living matter to be stored viably for future use. There are two main methods - slow freezing and vitrification. Slow freezing involves cooling at controlled rates while crystals form, while vitrification avoids ice crystal formation for small volumes by extremely fast cooling rates. Key aspects that determine cell survival include the cryoprotectants used, cooling and warming rates, and minimizing osmotic stress. Cryopreservation is important in reproductive medicine for gamete and embryo banking and has applications in cancer treatment.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    68 views50 pages

    Cryopreservation: Anuja Kamath, M.SC Bangalore Assisted Conception Centre

    Uploaded by

    Al Mamun
    Cryopreservation is the process of preserving living cells and tissues at low temperatures. It allows for cells, embryos, and other living matter to be stored viably for future use. There are two main methods - slow freezing and vitrification. Slow freezing involves cooling at controlled rates while crystals form, while vitrification avoids ice crystal formation for small volumes by extremely fast cooling rates. Key aspects that determine cell survival include the cryoprotectants used, cooling and warming rates, and minimizing osmotic stress. Cryopreservation is important in reproductive medicine for gamete and embryo banking and has applications in cancer treatment.

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    © All Rights Reserved
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    CRYOPRESERVATION

    ANUJA KAMATH, M.Sc


    BANGALORE ASSISTED CONCEPTION CENTRE
    Definitions
     Cryobiology- branch of biology that

    studies life at below normal temperature.

     Deals with effects of freezing and thawing

     Cells in “Suspended Animation”


    Definitions
     Cryopreservation -process of preserving and
    storing living system in a viable condition at
    low temperature for future use.

     Traditionally means freezing.

     Includes the ice free vitrification.


    Applications
     Most widely used in Reproductive Medicine

     Preservation of
    ◦ Gametes
    ◦ Embryos
    ◦ Gonadal Tissue.

     Forefront of research in ART .

     Tendency of IVF world to switch over to natural cycle IVF


    and SET.
    Cryopreservation in ART
    INDICATIONS-
     To gain time- postponement of fertility-

    cancer patient , OHSS


     To meet logistic needs-gamete or embryo

    donation
     Ensure backup-for repeat ET
    Aim of cryopreservation
    AIM
     To slow and/or shut down cellular activity
     To preserve structure

    Results:
     Variable depending on the complexity

    of the tissue to be preserved


    Water – In Living Tissues
     Major content of cell.
     universal compatible solvent.
     Unique properties for stability of living cells.
     Cooling – Induction of ice crystals.
     Phase transition from water to ice is the

    most profound challenge for survival.


     Volume expansion and Osmotic

    dehydration.
     Cellular Destruction – Intra cellular

    organelle & Phospholipid membrane.


    Freezing
     Two types
    ◦ Equilibrium cooling (Slow Freezing)
    slow-rate freezing, extracellular ice formation
    drives cellular dehydration through an equilibrium
    process.
    ◦ Non Equilibrium Cooling (Vitrification)
    form of rapid cooling, utilizes very high
    concentrations of cryoprotectant that solidify
    without forming ice crystals.
    Equilibrium Cooling
     Cryopreservation protocol

     Water content of a cell remains in osmotic


    balance with the surrounding solution during
    cooling

     In the presence of extra cellular ice crystals

     Slow cooling procedures


    Non-Equilibrium Cooling

     Cellular dehydration and cryoprotectant


    penetration are enforced by solutions with a
    high osmolarity before cooling commences

     Change very little during the cooling step

     All rapid-cooling procedures


    Seeding
     Manual or automated initiation of ice
    formation
     Usually as part of a slow cooling procedure
     Is initiated for example by touching the edge
    of the solution with a cold implement (e.g.
    forceps at -196°C)
     The cryoprotectant is seeded at a high
    subzero temperature (between - 4°C and -
    9°C) to initiate dehydration of the specimen
    Cryoprotectants
     Defined as any additive provided to cells
    before freezing and yields a higher post-thaw
    survival.
    Cryoprotectant
     protect cells from ice crystal damage by
    increasing solute
     Intracellular cyroprotectants (Permeating)

    ◦ 1,2 propanediol
    ◦ dimethyl sulfoxide DMSO
    ◦ ethylene glycol
    ◦ glycerol
     Extracellular cryoprotectants (Non
    Permeating)
    ◦ sucrose and raffinose
    Cryoprotectant
     Cryoprotectant - Non-permeating

     Any of a range of compounds which do not


    readily cross the cell membrane but which
    none the less provide cells with protection
    against cryoinjury

     The ones most commonly used for cells


    include sugars, polymers and proteins
    Cryoprotectant
     Cryoprotectant – Permeating

     Any of a range of low molecular weight


    hydrophilic compounds which can hydrogen bond
    with water

     DMSO, ethylene glycol, glycerol and propylene


    glycol

     Most cryoprotectants act as `antifreezes' in that


    they depress the ice nucleation temperature
    DMSO
     Antifreeze compound widely used for
    oocytes and embryos at a concentration of
    1.5 mol/l (11% v/v) for slow cooling, and
    >3.5 mol/l for rapid cooling

     It is commonly used in combination with


    glycol-type compounds as the combination
    interacts with water and biological materials
    in a slightly different way to glycols alone
    Ethylene glycol
     Also called 1,2-ethanediol
     Antifreeze compound widely used for

    oocytes and embryos


     It is commonly used in combination with

    DMSO
     It has relatively low toxicity
     Small size - penetrates membranes rapidly
    glycerol
     Also called 1,2,3-propanetriol
     Antifreeze compound widely used for sperm
    and blastocysts
     It has low toxicity, but is slow to cross
    membranes which can contribute to osmotic
    shock
    propanediol
     1,2-Propanediol
     Antifreeze compound - Used for human

    embryos
     It readily forms vitrifying solutions
     Commonly abbreviated as PG or PRO
     A potential problem is that one of its

    natural breakdown products is


    formaldehyde which has the potential to
    damage gametes
    sucrose
     A sugar – Disccharide

     Used to promote dehydration before, during


    and/or after cryopreservation

     Normally membrane impermeant and of low


    toxicity to oocytes and embryos
    trehalose
     A sugar – Disaccharide

     Used to promote dehydration before, during


    and/or after cryopreservation

     Normally membrane impermeant and of


    low toxicity to oocytes and embryos
    raffinose
     A sugar – Polysaccharide

     Used to promote dehydration before, during


    and/or after cryopreservation

     Normally does not cross the cell membrane


    (non-permeating/impermeant) and of low
    toxicity to oocytes and embryos
    Slow Cooling vs. Rapid Cooling
     Most dehydration and cryoprotectant
    permeation takes place before cooling
    commences

     Cooling is usually performed in a single step


    in which the specimen is cooled directly from
    a temperature >0°C to low subzero
    temperatures (<-130°C)
    Key aspects of
    cryopreservation
     Cryoprotectant used  Cryoprotectant used
     Seeding temperature  Vitrification temp.
     Freezing rate
     Warming rate
     Handling
     Thawing rate
     Data management
     Handling
     Data management

    Slow freezing vitrification


    Slow Cooling
     State during Cooling

     Ice present (usually seeded at -5 to -9°C)


     Cooling rate usually 0.3 to 1°C/min to
    between -30 and -80°C then >200°C/min

     State during Warming

     Ice present
     Warming rate – 4 to 100°C/min
    Vitrification
     State during Cooling
     Glass like state without ice formation
     Cooling rate usually 200 to 20,000°C/min
     Cooling rate depends on the type of container
    used

     State during Warming


     Maintains glass like, ice free state
     Warming rate depends on type of container
     Warming strategy usually 300 to >12
    000°C/min
    Slow Cooling Vitrification

    Slow Fast

    Programmable freezer No equipment

    Less skill Technically demanding

    Low concentration of CPA High concentration of CPA

    Seeding No seeding

    Not useful for oocytes Can be used for oocytes – No


    meiotic spindle damage
    Ice formation No Ice formation
    Rate of cooling - 0.3 to 1°C/min to Rate of cooling - 200 to
    between -30 and -80°C then 20,000°C/min
    >200°C/min
    No glass like state Glass like state

    Cooling-Independent on type of Cooling depends on type of


    container container
    Less efficient More efficient

    Warming rate – 4 to 100°C/min Warming rate -300 to


    >12,000°C/min

    Warming – Ice formation Warming – Glass like state

    Less CPA Injury More CPA Injury

    Less Osmotic Shock More osmotic shock


    Cells – Less fragile Cells – More fragile

    More membrane stability Less membrane stability

    More exposure to CPA Less exposure to CPA

    Not useful for small volume Very useful for small volume

    Strict Control – Not required Strict Control - Required

    Long equilibration time Short equilibration time


    Safety Issues in Cryopreservation
     Skin frost bite

     Explosion of cryovials – During warming

     Large amount of LN2- O2 deprivation

     Cross Infection –Viral Infections (Storage in


    vapor phase will reduce the chance of viral
    contamination)
    Oocyte freezing
     Denuded MII oocytes are frozen.
     Oocyte is a single cell, water content more
     Damage to oocyte during freeze thaw cycle
    more
     Slow freezing and vitrification
    Slow freezing of Oocyte
     Oocyte freezing kit
     60 mm petri dish
     Oocyte freezing straw
     170 micron pipette
     Freezer
     Forceps
    Freezing medium
     Vial 1 : phosphate-buffered saline (PBS)

    (WASH)

     Vial 2 : 1.5M 1,2-PROH

     Vial 3 : 1.5M 1,2-PROH + 0.3M sucrose


    Slow freezing of Oocyte
    1. Warm to room temperature for minimum 30 minutes.
    2. Wash the oocytes in 3 ml medium from Vial 1 at room temperature.
    3. Transfer the oocytes to 0.5 ml medium from Vial 2 for 10 minutes at
    room temperature.
    4. Transfer the oocytes to 0.5 ml medium from Vial 3 and rapidly load
    into straws using this medium as transfer medium.
    5. Transfer the straws to the controlled rate freezer and cool from room
    temperature to -7ºC at a rate of 2ºC per minute.
    6. Seed manually at -7°C.
     When the solution is white, seeding has been initiated.

    7. Hold for 10 minutes at -7°C


    8. Cool from –7ºC to –30ºC at a rate of 0.3ºC per minute.
    9. Cool from –30ºC to –150ºC at a rate of 50ºC per minute.
    10. After 10-12 minutes of temperature stabilisation, transfer the
    straws into liquid nitrogen and store at –196ºC.
     Load the straw(s) with oocytes inthe following
    order:
    a. 1-inch medium
    b. 1/4-inch air space
    c. 1/2-inch medium
    d. 1-inch medium containing oocytes
    e. 1/4-inch air space
    f. Fill the remainder of the straw with medium.
    Make sure the medium completely wets the straw
    sealant at the end of the straw to ensure a proper
    seal.
    Thawing of Oocyte
     Thawing medium

    ◦ VIAL 1 : 1M PROH + 0.3M SUCROSE

    ◦ VIAL 2 : 0.5M PROH + 0.3M SUCROSE

    ◦ VIAL 3 : 0.3 M SUCROSE

    ◦ VIAL 4 : PBS
     Warm for a minimum of 30 minutes at room temperature.
     Remove straws from liquid nitrogen and keep at room temperature

    for 30 seconds. Then place straws in water at 30°C for 40 seconds


    until all traces of ice have disappeared.
     Open the straws or cryo-tubes according to the manufacturer’s

    instructions and expel the contents into a Petri dish.


     Place the oocytes in medium from Vial 1 at room temperature for 5

    minutes.
     Transfer oocytes into medium from Vial 2 at room temperature for

    5 minutes and then into medium from Vial 3 at room temperature


    for 10 minutes.
     Transfer oocytes into medium from Vial 4 for 10 minutes at room

    temperature and for an additional 10 minutes at 37°C.


     Place the oocytes in culture medium in CO 2 at 37°C for a minimum

    of 2 hours before fertilisation.


    Embryo freezing and thawing
     Embryo freezing medium
    ◦ Vial 1 : HEPES buffered medium
    ◦ Vial 2 : 1.5 M PROH
    ◦ Vial 3 : 1.5 M PROH + 0.1 M Sucrose

     Embryo Thawing Medium


    ◦ Vial 1 : 1 M PROH + 0.2 M Sucrose
    ◦ Vial 2 : 0.5 M PROH + 0.2 M Sucrose
    ◦ Vial 3 : 0.2 M Sucrose
    ◦ Vial 4 : HEPES Buffered Medium.
    Blastocyst Freezing and Thawing
     Blastocyst freezing medium
    ◦ Vial 1 : HEPES buffered media
    ◦ Vial 2 : 5% glycerol
    ◦ Vial 3 : 9% glycerol +0.2 M sucrose

     Blastocyst thawing medium


    ◦ Vial 1 : 0.5 M Sucrose
    ◦ Vial 2 : 0.2 M Sucrose
    ◦ Vial 3 : HEPES buffered media
    vitrification
     Vitrification cooling medium
    ◦ Vial 1 : Buffered Medium

    ◦ Vial 2 : Ethylene glycol

    ◦ Vial 3 : Ethylene glycol, propanediol, sucrose


     Take 0.5 ml of each media in each well of a 4
    well dish, Warm 37 deg C, for 30 mins.
     Select the embryos and transfer them to well

    1 for 5 – 10 mins.
     Transfer the embryos to well 2 for 1 ½ min.
     Finally transfer the embryos to well 3 for 30

    sec and load the cryodevice.


    Vitrification warming
     Warm 0.5 ml of all media at 37 deg C
     Take out the cyodevice from canister into

    container with LN2.


     Open the cap of the device and plunge it in

    well 1 for 30 sec


     Gather the embryo and transfer to well 2 for

    1 min
     Move the embryo to well 3 for 2 min.
     Move the embryo to well 4 for 5 mins.
     Transfer to pre equilibrated culture dish.
    Cryodevices

     McGill Cryoleaf
     Rapid i
     cryotop
     Cryolock
     Should be performed by trained embryologist
     Embryos should be loaded on the device and
    plunged in LN2 within 30 secs
     Volume should be minimum
     First step in thawing very important.
     Temp should be maintained at 37 deg while
    thawing.
    Thank you

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