Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

ELS/ALS Exam Review > Cryopreservation > Flashcards

Cryopreservation Flashcards

1
Q

What are two reasons for cryopreservation’s cell-specific nature?

A
  1. Dependent on the amount of intracellular water

2. Dependent on the type of cryoprotective agent (CPA) used

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What does “CPA” stand for?

A

Cryo-Protective Agents

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What are the four factors used to characterize CPA’s?

A
  1. Relatively low molecular weight
  2. High solubility in water
  3. Cell permeability properties
  4. Non-toxic properties at high intracellular concentrations
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

List six reasons why spermatozoa are ideal cells to cryopreserve.

A
  1. Smallest human cell
  2. Relatively small volume
  3. Large surface area
  4. Very little cytoplasm
  5. Less total intracellular water than many other cells
  6. Exist individually which allows for efficient dehydration
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

List six reasons why spermatozoa are ideal cells to cryopreserve.

A
  1. Smallest human cell
  2. Relatively small volume
  3. Large surface area
  4. Very little cytoplasm
  5. Less total intracellular water than many other cells
  6. Exist individually which allows for efficient dehydration
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

When was the first successful cryopreservation of sperm and what CPA was used?

A

1953; glycerol

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What is the most widely used CPA for freezing human spermatozoa?

A

Glycerol

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is the surface area of a typical oocyte?

A

4.5 x 10^4 micrometers^2

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What is the surface area of a typical sperm cell?

A

120 micrometers^2

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What is the volume of a typical oocyte?

A

9 x 10^5 micrometers^3

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What is the volume of a typical sperm cell?

A

28 micrometers^3

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Compare the ratio of surface area to volume between oocytes and sperm

A

Oocyte: 0.05

Sperm 4.3

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What are the principles of cryopreservation based on?

A

The laws of thermodynamics

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Name three ways in which cryopreserving human sperm without a CPA subjects the cells to damage.

A
  1. Plasma membrane swelling (water expands during freezing)
  2. Acrosomal leakage
  3. Breakdown
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What are the two functions of glycerol in the cryopreservation of human sperm?

A
  1. Remove or reduce water content of cells

2. Minimize intracellular ice formation during freezing

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Why do sperm cells initially shrink when placed in CPA’s?

A

Because of a reduction in intracellular water due to increased extracellular osmolarity

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

How do sperm cells eventually return to their (almost) original volume with CPA’s?

A

Because osmotic equilibrium is eventually reached through the CPA penetrating the cell and displacing the intracellular water of the sperm

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

The addition of a CPA is usually accompanied by what?

A

Reduction in Temperature

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

As the cooling process continues during the cryopreservation of sperm, within what T range does extracellular ice formation occur?

A

-5 degrees C to -15 degrees C

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

What is associated with the extracellular ice formation phase during the cryopreservation of human sperm?

A

An extracellular solid phase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

During cryopreservation, what happens to the inside of the sperm cell?

A

It remains unfrozen but supercooled.

22
Q

Describe the chemical potential and osmotic behavior of supercooled intracellular water during sperm cryopreservation.

A

It has a higher chemical potential and diffuses out of the cell osmotically.

23
Q

Define “hypertonicity” (of a solution).

A

having a higher osmotic pressure than that of a specified physiological solution

24
Q

Why does sufficient time need to be allowed during sperm cryopreservation?

A

Because freezing continues extracellularly, resulting in hypertonicity and a further reduction of water from sperm cells, leading to almost complete dehydration, so for this process to complete, it takes time to reach that kind of osmotic equilibrium

25
Q

How does the sperm thaw process work on a cellular level?

A

The reverse of the freezing process

26
Q

Compare the warming process of sperm cells that have been frozen slowly vs. rapidly.

A

Cells that have been frozen slowly must be thawed slowly, whereas cells that have been frozen rapidly should be thawed rapidly.

27
Q

Glycerol is a standard CPA for human spermatozoa at what concentration?

A

5% to 10% v/v

28
Q

How can cryosurvival rates of human sperm with glycerol improve?

A

By adding more complex buffer medium extenders

29
Q

What are the nine complex buffer medium extenders used as sperm cryoprotectants?

A
  1. Glycerol
  2. Citrate-egg yolk-glycerol
  3. TEST-C-I
  4. TEST-C-II
  5. TEST-C-III
  6. TEST-milk
  7. HSPM (Human Sperm Preservation Medium)
  8. HEPES-KOH-G
  9. Egg yolk-free
30
Q

What is the composition of HSPM and why is it used as a sperm cryoprotectant?

A

A modified Tyrode’s medium with 7.5% glycerol and has been reported to yield a higher post-thaw recovery of motile sperm than egg-yolk containing media with glycerol.

31
Q

What is the composition of IUI-ready cryoprotectant and why is it used as a sperm cryoprotectant?

A

HEPES-buffered human tubal fluid with 1% human serum albumin, 4% sucrose and 6% glycerol for semen samples preprocessed using gradient density centrifugation; recovery of motile sperm was comparable to conventional semen cryopreservation using TEST yolk buffer with glycerol, followed by density gradient centrifugation.

32
Q

How is glycerol alone used as a sperm cryoprotectant?

A

Added to near semen to a final concentration of 7.5%v/v

33
Q

How is the sperm cryoprotectant Citrate-egg yolk-glycerol prepared (three steps)?

A
  1. 353mOsm sodium citrate, pH 7.0, 20% v/v fresh egg yolk, 2% v/v 325 most fructose.
  2. Mixture is centrifuged at 10,000g for 10 minutes and the supernatant is filtered through filter paper.
  3. Glycerol is added to a final concentration of 7.5% v/v..
34
Q

What is the composition of the sperm cryoprotectant TEST-C-I and how is it prepared?

A

A zwitterionic buffer system prepared by titrating 325 most TES with most TRIS to make the TEST solution; 48% v/v TEST, 30% sodium citrate, 20% egg yolk, 2% fructose are mixed and processed as described for CEG; Glycerol is added to a final concentration of 6%.

35
Q

What is the composition of the sperm cryoprotectant TEST-C-II and how is it prepared?

A

70% v/v TEST, 12.5% sodium citrate, 17.5% egg yolk, mixed and processed as CEG only glycerol is added to a final concentration of 10%

36
Q

What is the composition of the sperm cryoprotectant TEST-C-III?

A

A solution of TEST-C-II but WITHOUT glycerol

37
Q

What is the composition of the sperm cryoprotectant TEST-milk and how is it prepared?

A

30% by volume TEST, 30% most powdered milk, 18% sodium citrate, 20% egg yolk, 2% fructose; Mixture is heated to 92-95 degrees C for 10 minutes, filtered through sterile gauze and centrifuged at 10,000g for 15 minutes; Supernatant is filtered through filter paper and osmotic pressure is adjusted to 325mOsm and pH to 7.0; Glycerol is added to a final concentration of 15%.

38
Q

Out of eight sperm cryoprotectant extenders, which two were superior in maintaining sperm viability?

A

Both a combination of zwitterions TES and TRIS with sodium citrate and egg yolk

39
Q

Cryosurvival rates of human sperm improved with the addition of glycerol to what?

A

Complex buffer medium extenders

40
Q

Why are zwitterion buffers suspected to improve in the recovery of motile sperm post-warming?

A

Attributed to the zwitterions’ abilities to bind free hydrogen and hydroxyl ions in the surrounding medium, aiding in the dehydration process of the sperm

41
Q

What are five reasons that egg yolk is used in sperm cryopreservation?

A
  1. Studies show that it helps maintain sperm viability;
  2. Improves membrane fluidity;
  3. Also studies show higher sperm longevity,
  4. Higher percent survival
  5. As well as the ability to penetrate cervical mucus post-thaw.
42
Q

What is the composition of the sperm cryoprotectant HSPM?

A

Human sperm medium; a modified Tyrode’s media with glycerol added to a final concentration of 15%

43
Q

How is the sperm cryoprotectant HEPES-KOH-G prepared?

A

325mOsm HEPES is titrated with 325mOsm KOH to pH 7.0, 40% HEPES KOH, 40% 325mOsm glucose and 20% egg yolk are mixed and processed as described for CEG; Glycerol is added to a final concentration of 6%.

44
Q

What is the composition of egg-yolk free sperm cryoprotectant?

A

Buffered medium containing human serum albumin (HSA) and glycerol

45
Q

What is the difference between thawing slow-freeze vs. vitrified samples of ejaculated, epididymal, and testicular sperm?

A

Slowly at ambient T for 30-60 minutes vs. rapidly thawed in a 37 degrees C water bath for 5-10 minutes, respectively

46
Q

Define “Client Depositor.”

A

An individual who banks his semen for deferred insemination of a sexually intimate partner or simply for long-term storage without a designated plan for use. He is not a donor at the time of semen cryopreservation.

47
Q

What five comprehensive histories are taken from potential sperm donors and how long must these date back to?

A
  1. Medical history
  2. Social history
  3. Sexual history
  4. Family medical history
  5. Genetic history;
    Minimum of two generations
48
Q

Guidelines exist for what six aspects of semen donation?

A
  1. Retrieval
  2. Processing
  3. Preservation
  4. Storage
  5. Labeling
  6. Distribution
    (of semen samples)
49
Q

What two organizations have established relevant standards for semen donation and banking?

A
  1. The American Association of Tissue Banks (AATB)

2. American Society for Reproductive Medicine (ASRM)

50
Q

In NY, records of donor semen us that resulted in pregnancy must be kept for at least how many years after semen distribution?

A

25 years

51
Q

What federal organization has published regulations for semen banking?

A

FDA

ELS/ALS Exam Review (7 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions