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Questions and Answers
What are mesenchymal stem cells (MSCs) primarily known for in regenerative medicine?
What are mesenchymal stem cells (MSCs) primarily known for in regenerative medicine?
How many mesenchymal stem cells are required per kg of body weight for transplantation?
How many mesenchymal stem cells are required per kg of body weight for transplantation?
What is the main purpose of cryopreservation in the context of MSC storage?
What is the main purpose of cryopreservation in the context of MSC storage?
Which cryopreservation method involves uncontrolled freezing?
Which cryopreservation method involves uncontrolled freezing?
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What do both slow freezing and vitrification methods require to improve cell survival rates after thawing?
What do both slow freezing and vitrification methods require to improve cell survival rates after thawing?
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What characterizes the vitrification method in cryopreservation?
What characterizes the vitrification method in cryopreservation?
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What is a common outcome desired after thawing cells that have undergone cryopreservation?
What is a common outcome desired after thawing cells that have undergone cryopreservation?
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What is the first temperature stage in the three-step freezing method for cells?
What is the first temperature stage in the three-step freezing method for cells?
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What is the purpose of adding cooled cryoprotective agents (CPAs) during the freezing process?
What is the purpose of adding cooled cryoprotective agents (CPAs) during the freezing process?
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How long should cells be stored at -20 ℃ in the three-step freezing method?
How long should cells be stored at -20 ℃ in the three-step freezing method?
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What is the final storage temperature for long-term preservation of cells?
What is the final storage temperature for long-term preservation of cells?
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What is a key characteristic of the vitrification method?
What is a key characteristic of the vitrification method?
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What concentration range of CPAs is used in the vitrification method?
What concentration range of CPAs is used in the vitrification method?
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Which of the following actions is NOT part of the three-step freezing method?
Which of the following actions is NOT part of the three-step freezing method?
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What potential benefit does induced ice nucleation provide during the freezing process?
What potential benefit does induced ice nucleation provide during the freezing process?
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What is typically the duration for storing cells at -85 ℃ within the three-step freezing method?
What is typically the duration for storing cells at -85 ℃ within the three-step freezing method?
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In the vitrification process, which state do cell suspensions transition to rapidly?
In the vitrification process, which state do cell suspensions transition to rapidly?
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What is the primary function of cryoprotective agents (CPAs) during the cryopreservation process?
What is the primary function of cryoprotective agents (CPAs) during the cryopreservation process?
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Which of the following is NOT a common penetrating cryoprotectant?
Which of the following is NOT a common penetrating cryoprotectant?
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What is the effect of low cooling rates in the slow-freezing method?
What is the effect of low cooling rates in the slow-freezing method?
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How do CPAs help in managing intracellular ice formation during cryopreservation?
How do CPAs help in managing intracellular ice formation during cryopreservation?
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Which mechanism occurs as CPAs are added before cryopreservation?
Which mechanism occurs as CPAs are added before cryopreservation?
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What happens during fast cooling in the context of cryopreservation?
What happens during fast cooling in the context of cryopreservation?
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What is a common property of non-membrane permeating cryoprotectants?
What is a common property of non-membrane permeating cryoprotectants?
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In the context of cryopreservation, what should be the condition of the cell to reduce dehydration injury?
In the context of cryopreservation, what should be the condition of the cell to reduce dehydration injury?
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What is the result of using too high a concentration of CPAs during the slow-freezing process?
What is the result of using too high a concentration of CPAs during the slow-freezing process?
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Why is cryopreservation considered a promising method for the storage of transplantable cells and tissues?
Why is cryopreservation considered a promising method for the storage of transplantable cells and tissues?
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What is the purpose of using hydrogels or alginate capsules in the context of mesenchymal stem cells (MSCs)?
What is the purpose of using hydrogels or alginate capsules in the context of mesenchymal stem cells (MSCs)?
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Which of the following is a major consideration when assessing the viability of spheroids after rewarming?
Which of the following is a major consideration when assessing the viability of spheroids after rewarming?
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What aspect of MSC storage has been a focus of investigation in recent years?
What aspect of MSC storage has been a focus of investigation in recent years?
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What is the main limitation associated with traditional cryopreservation techniques?
What is the main limitation associated with traditional cryopreservation techniques?
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What is the primary advantage of vitrification compared to slow freezing in cryopreservation?
What is the primary advantage of vitrification compared to slow freezing in cryopreservation?
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What is a common drawback associated with the use of high concentrations of CPAs in vitrification?
What is a common drawback associated with the use of high concentrations of CPAs in vitrification?
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Which method demonstrates excessive cell death primarily in the core region after the freezing process?
Which method demonstrates excessive cell death primarily in the core region after the freezing process?
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What is the key mechanism by which CPAs increase viscosity during the process?
What is the key mechanism by which CPAs increase viscosity during the process?
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What is the recommended rate of freezing when using liquid nitrogen for vitrification?
What is the recommended rate of freezing when using liquid nitrogen for vitrification?
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Which of the following statements is true regarding the difference between equilibrium and non-equilibrium vitrification?
Which of the following statements is true regarding the difference between equilibrium and non-equilibrium vitrification?
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How does slow freezing compare to vitrification in terms of sample handling and equipment requirements?
How does slow freezing compare to vitrification in terms of sample handling and equipment requirements?
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What cellular outcome was observed with vitrification that was more favorable than that of slow freezing?
What cellular outcome was observed with vitrification that was more favorable than that of slow freezing?
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What effect does hydrating CPAs have on sample viscosity during the vitrification process?
What effect does hydrating CPAs have on sample viscosity during the vitrification process?
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Why might CPAs be challenging to manage during the vitrification process?
Why might CPAs be challenging to manage during the vitrification process?
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Study Notes
Stem Cell Cryopreservation
- Mesenchymal stem cells (MSCs) are multipotent stem cells with potential in regenerative medicine and tissue engineering
- MSCs regulate the immune system and have self-renewal and multi-lineage differentiation potential in vitro
- 1x106 cells per kg of body weight are needed for MSC transplantation, to increase and cultivate MSCs from various donors, and for long-term storage
- Cryopreservation is a method to preserve the structural and functional integrity of cells and tissues at low temperatures for long periods
- Two main forms of MSC cryopreservation are slow freezing (uncontrolled freezing) and vitrification (controlled freezing)
- Both methods use cryoprotectants (CPAs) which are additives that increase the chance of cell survival after thawing
Cryoprotectants (CPAs)
- CPAs pass through the plasma membrane and form hydrogen bonds with water
- They reduce osmotic pressure, reducing intracellular ice formation
- Two types of cryoprotectants:
- Cell membrane-permeating: glycerol (GLY), dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG)
- Non membrane-permeating: polymeric materials (polyvinyl pyrrolidone, hydroxyethyl starch (HES)), and small molecules (trehalose)
Slow Freezing
- Cooling rate is low enough to dehydrate cells and prevent intracellular ice formation
- Uses low concentrations of CPAs for optimal dehydration
- This method involves gradually decreasing the temperature to maintain optimal cell structure in a controlled rate
- CPAs are added before cryopreservation to increase solute concentration extracellularly and cause the cells to shrink in response to osmotic pressure
- Critical to maintain a controlled rate of cooling
- Slow cooling prevents irreversible dehydration and damage to cell structures while fast cooling leads to ice nucleation and potentially cell lysis
Vitrification
- A rapid freezing method that transforms cell suspensions directly into a glass state, avoiding ice formation
- High concentration of CPAs are used to increase viscosity of the sample and prevent ice crystal formation through strong hydration and interactions
- Low temperature liquid nitrogen allows for rapid freezing
- Two forms of vitrification: Equilibrium and Non-equilibrium vitrification
- Equilibrium vitrification involves specific formulations of CPAs in the mixtures put into cells before direct submersion into liquid nitrogen to reach osmotic equilibrium
- Non-equilibrium vitrification involves high concentration of CPAs that rapidly penetrate cells immediately placed into liquid nitrogen
Comparison of Methods
- Slow Freezing: Advantages: low CPA concentrations, simple handling, large volume storage potential, and preservation of tissue structures, Disadvantages: ice formation, potential structural damage
- Vitrification: Advantages: Prevents ice formation and preserves tissue structure, Disadvantages: high CPA concentrations, CPA-related toxicity, complex CPA addition and removal, difficult handling
Applications
- MSCs are encapsulated in hydrogels or alginate capsules to mimic tissue and organ structure to facilitate transplantation
- Cryopreservation is one of the critical processes in cell storage and long-term preservation (important for both quantity and quality)
Additional Notes
- Lower temperatures reduce CPA diffusion rate into and out of cells, reducing osmotic shock, while maintaining the stability of cell membranes
- Lower temperatures protect cell membranes, reduce the impact of CPA on cell membranes, and increase solution viscosity thus reducing cell damage
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Description
Test your knowledge on the cryopreservation of mesenchymal stem cells (MSCs) and their relevance in regenerative medicine. This quiz covers key concepts like cryoprotectants, freezing methods, and the importance of MSCs in tissue engineering. Explore the different techniques and understand how they impact cell viability.
