Cryogenic Biotechnology

Question 1

Normal Air is what kPa?

Answer 1

~21 kPa O2 (101 kPa = 1 atm)

Question 2

Mild Hypoxia is what kPa?

Answer 2

0.25-1.0 kPa O2

Question 3

Intermediate hypoxia is what kPa?

Answer 3

.001-0.25 kPa O2

Question 4

During mild hypoxia what are 3 things involved with this?

Answer 4

• HIF-1 (Hypoxia Inducible Factor-1)
• Anaerobic energy production genes up-regulated: glycolytic enzymes, glucose transporters
• Antioxidant genes upregulated: catalase, superoxide dismutase

Question 5

What is Anoxia kPa>? What occurs during Anoxia?

Answer 5

• <0.001 kPa O2
• HIF-1 response alone is not sufficient to protect the cells from free radicals
• Oxidative phosphorylation ceases
• ATP demand must also decrease
• Cells of some organisms enter into a state called “suspended animation”

Question 6

What occurs during Suspended animation?

Answer 6

• No observable movement is visible
• Developmental progression ceases

Question 7

What happens to Turtle hepatocytes during Suspended Animation?

Answer 7

• Turtle hepatocytes in this state are capable of reducing their ATP demand by 94%
• limit protein synthesis, ion channel
  activity, and anabolic pathways

Question 8

What happens to Zebrafish embryos and C. elegans embryos during Suspended Animation

Answer 8

Zebrafish embryos and C. elegans embryos stop moving and developing

Question 9

Carbon Monoxide is a competitive inhibitor of oxygen binding. What does this cause?

Answer 9

• Carbon Monoxide binds and inhibits cytochrome c oxidase (complex IV), causing free radical production
• Thus Carbon Monoxide effectively lowers the amount of oxygen electron carriers

Question 10

What did we learn about C. elegans from the different treatments in anoxia and hypoxia?

Answer 10

• C. elegans embryos are capable of surviving in anoxia and mild hypoxia, but intermediate hypoxic conditions are lethal
• CO induced suspended animation in embryos experiencing intermediate hypoxia
• Embryos that had entered CO induced suspended animation were highly viable

Question 11

Cryogenic Medicine exists. What are some examples

Answer 11

• Cooling of vital organs
• Particularly heart and brain to reduce the metabolic rate
• Lowers oxygen consumption
• Protect from apoptosis
• Only need to reduce 3-4ºC for these effects

Question 12

Provide some actual examples of Cryogenic Medicine

Answer 12

• The Arctic Sun
• Nasal Cooling
• Catheters

Question 13

What are the treatment stages in order of Cryotherapy with Liquid Nitrogen

Answer 13

1. Application
2. Blister stage
3. Crusting stage
4. Clearance

Question 14

Who was the first person cryogenically frozen?

Answer 14

73-year-old psychologist Dr. James Bedford in 1967

Question 15

Long Answer: How is Cryonics performed?

Answer 15

• After official death, body is packed into ice and injected with heparin to prevent blood clotting during transportation
• After arrival at the facility, blood is replaced with cryoprotectant solution to perform vitrification
• Completing the vitrification (deep cooling without freezing) the body is cooled on a bed of dry ice until it reaches -130°C
• After the vitrification process the body is inserted into a container filled with liquid nitrogen at a temperature of -196°C

Question 16

What is Cryobiology?

Answer 16

Study of biological materials or systems at temperatures below normal

Question 17

Define Suspended Animation

Answer 17

• Stop of life process
• metabolism, cellular activity, body temperature, respiration and heartbeat decrease, the oxygen is cut off from the body, and the cells stop to divide yet they are still alive

Question 18

Organisms such as frogs can survive winter in a completely frozen state – How?

Answer 18

Tundra frogs secrete urea (from urine) into interstitial fluids and high glucose concentration into the bloodstream to serve as cryoprotectants (to prevent freeze-thaw damage).

Question 19

Explain Cryopreservation

Answer 19

• long term storage of cells, tissues, cultures, gametes, embryos, organs for transplantation
• liquid nitrogen (–196°C), –80°C freezer

Question 20

What are some Freezable tissues?

Answer 20

• Blood (special cells for transfusion, or stem cells)
• Tissue samples like tumors and histological cross sections
• Human spermatic cells
• Human embryos that are 2, 4, or 8 cells when frozen (pregnancies have been reported from embryos stored for 9 years).

Question 21

Define Lyophilization

Answer 21

• aka Freeze-drying
• dehydration process used to preserve a perishable material or make the material more convenient for transport

Question 22

Define Cryosurgery

Answer 22

destruction of unhealthy tissues using cryogenic gases or liquids (wart removal, cervical disorders, prostate cancer)

Question 23

What is the effect of low temperatures on the cells?

Answer 23

• survival of the cell depends on
  water content
• the most resistant to cold: seeds, spores
• very susceptible to cold: animal cells
• mechanical damage of biomembranes by ice crystals
• optimalization of the procedure needed – different kinds of material require different speed of deep freezing and defrosting

Question 24

What are the 4 effects of low temperatures on the cells

Answer 24

1. Solution effect solutes are excluded from the crystal structure of the ice. High salt concentrations can be very damaging.
2. Extracellular ice formation - cause
   mechanical damage due to crushing
3. Dehydration due to extracellular ice formation
4. Intracellular ice formation

Question 25

What are some examples of cryoprotective substances and what do they do?

Answer 25

- Ex.) Glycerol, PEG, DMSO - lower solidification temperature, partial water replacement in the cell, reduction of ice formation

Question 26

What does quick freezing (using liquid nitrogen, helium) – vitrification of water mean?

Answer 26

solidification without any crystal ice formation, or formation of very small crystals which does not lead to significant damage of the cell

Question 27

What are 3 assessments of viability after deep freezing?

Answer 27

A. cultivation method – is the cell able to produce offspring? - cells are cultivated on plates and colonies counted – CFA (colony forming assay) B. membrane integrity assessment – is the cell still protected by membrane barrier? - microscopic inspection of dye staining - intact membrane does not allow penetration of dye inside cells C. metabolic activity testing – MTT testing - based on measurements of mitochondrial dehydrogenase activity

Question 28

What is Vitrification?

Answer 28

- a process of converting aqueous solutions into the glassy (amorphous) state

Question 29

How is the formation of ice prevented by the vitrification process?

Answer 29

- control of cooling rate - the introduction of some agents that suppress the formation of ice

Question 30

Define Cryoprotectants

Answer 30

Additives, which are used in cryobiology and cryonics or produced naturally by organisms

Question 31

Polar fish and insects make “____” proteins (ice blockers) which prevent what?

Answer 31

- Antifreeze - the nucleation of ice ; - the growth of the already formed ice below its freezing temperature

Question 32

How to prevent the formation of ice?

Answer 32

- Glycerol and sugar glucose are natural cryoprotectants. - There several synthetic cryoprotectants. - Concentrated cryoprotectants are toxic.

Question 33

How to escape the devitrification problem (thermal damage)?

Answer 33

Efficient and nontoxic ice blockers, for example, synthetic anti-freeze proteins, will strongly reduce the mechanical damages produces by the devitrification problem in the living tissue.

Question 34

14 stages of Vitrification in Biological systems

Answer 34

1) Living tissue contains much of water 2) Water is component of a cellular solution in living tissue 3) When tissue is cooled below freezing temperature, water molecules gather together and form growing ice crystals 4) Growing ice expel other molecules from the ice lattice to form a harmful concentrated solution 5) On a cellular scale, ice forms first outside cells 6) Growing ice causes cells to dehydrate and shrink 7) Finally cells are left damaged and squashed between ice crystals. The damage is mostly mechanical 8) Adding cryoprotectants to cellular solution can prevent crystallization of water to ice 9) Instead of freezing, molecules just move slower and slower as they are cooled 10) Finally, at very low temperature, molecules become locked in place and an amorphous solid is formed. Water that becomes solid without freezing is said to be "vitrified": 11) Cryoprotectants are added to biological system before deep cooling 12) There is no damage to cells during cooling because no ice is formed 13) Finally cells are vitrified and biological time is stopped 14) Because no ice is formed, vitrification can solidify tissue without structural damage