Cryopreservation of cells is a cell storage procedure in which specific cells are cooled to sub-zero temperatures to be thawed subsequently for use at some other time with some level of efficacy. This technique is essential for numerous purposes, especially in the medical field, as well as disease treatment and therapeutic areas.
As the human population continues to progress into the twenty-first century, technologies of cell preservation have strategically engaged in enhancing viability of recovering the cells, protecting the cells from damage and simplifying the process.
The preservation of oocytes and embryos using vitrification, which stops the development of ice crystals by quickly chilling cells to a glass-like condition, has gained popularity.
Indications
Stem Cell Therapy: Â Cells such as hematopoietic stem cells from bone marrow or umbilical cord blood are stored and later used to treat leukemia or lymphoma.
Regenerative Medicine: The cells which are employed in regenerative medicine such as mesenchymal stem cells or adipose derived stem cells are stored for future utility.
Cell-based Vaccines: It is also possible to make some vaccines from cell lines that are either stored or lyophilized so that they can be made to release the vaccine later.
Research: Cryopreserved cell lines are used in different research activities, such as in the toxicity and efficacy of drugs, genetic disorders, and carcinogenesis.
Fertility Preservation: Cells that can be frozen include sperm, eggs, and embryos for patients who might require treatment that can affect their fertility, like chemotherapy.
Tissue Engineering: From the cell sources to generate tissue construct or organ replacement, the cells are banked frozen until they are required for implantation or other processing.
Contraindications
Autoimmune Conditions: When cryopreserved cells are introduced into a patient with a severe autoimmune illness, the immune response may be adversely impacted.
Allergies and Reactions: Allergies of very high severity to the cryopreservation agents or media used could be a threat.
Health Conditions: Contraindications may include severe systemic health conditions or conditions that hamper the ability of the individual’s cells to take the therapies (severe liver or kidney disease, for instance).
Outcomes
Equipment
Cryopreservation Vials
Cryogenic Freezers
Liquid Nitrogen Storage Tanks
Cryo Boxes
Cryoprotectants
Thawing Equipment
Cryogenic Labels and Tracking Systems
Patient Preparation
Pre-Procedure Consultation: Patient assessment and making sure there are consent from the patient about their history.
Pre-Medication: Prescribe pre-medication if needed, this would be in cases where the anesthetic may cause an allergic reaction such as giving antihistamines or corticosteroids.
Thawing Cells: Thaw the cells by warming immediately the cryopreserved cell suspension in the water bath at 37°C. Avoid overheating.
Cell Washing: Gently transfer cells to a sterile centrifuge tube and wash them with an appropriate medium to remove cryoprotectants.
Patient Preparation: Ensure the patient is properly hydrated and has adequate venous access if needed for cell infusion.
Infusion: Then, procedure to add the thawed cell suspension into the patient based on the determined flow sheet, often over a period to avert adverse reactions in the patient.
Technique
Step 1-Preparation:
Cell Culture: Inoculate the flask with appropriate media to the value of the desired density. Often, cells are collected in the logarithmic growth phase to increase the chances of the harvested cells’ survival.
Equipment: Familiarize yourself with requirements about the cleanliness of the work environment like laminar flow hood and obtainable apparatus; the centrifuge, the cryovials, and the programmable freezer or cryopreservation unit.
Step 2-Harvesting Cells:
Detach Cells: For adherent cells, it is necessary to use the cell scraper or trypsin to make the cells release from the culture surface.
Wash Cells: Centrifuge the cell suspension, as this would help eliminate the culture medium and trypsin. Wash with an appropriate buffer or sequential media.
Count Cells: You should use the hemocytometer or an automated cell counter to measure the cell density.
Step 3-Prepare Cryoprotectant Solution:
Cryoprotectants: Some of the most frequently employed cryoprotective are DMSO or glycerol. Prepare the cryoprotective agent which might be DMSO (dimethyl sulfoxide) or glycerol at a concentration of 10% in either the balanced medium or serum.
Mix: Do not allow the cryoprotectant to encounter the cells before being well mixed with the cell suspension as this may cause more harm to the cells.
Step 4-Resuspend Cells:
Combine: Centrifuge & dilute the cells with the cryoprotectant solution. The general proportion is 1:1 commonly termed as a cell pellet: Cryoprotective solution ratio based on cell type and concentration.
Aliquot: Dispense the cell suspension into cryovials, usually 1-2 million cells per vial.
Step 5-Freezing:
Controlled-Rate Freezing: It is frozen at a controlled rate with a controlled rate freezer then stored at a very low temperature. This assists in avoiding the development of ice crystals that can be destructive to cells. The freezing rate is usually set at 1°C per minute at -80°C.
Step 6-Storage:
Liquid Nitrogen: After that, move the cryovials to another vial shredding container containing liquid nitrogen and store them for long term. This makes sure that the cells are well preserved at -196°C thus stopping any biological activities and retaining the functionality of the cells.
Step7-Thawing:
Warm-Up: Cryovials should be quickly thawed in a 37°C water bath with mild shaking until only a few ice crystals are visible.
Dilution: To reduce toxicity, this procedure must be done quickly, and after that, the cells should be put into the culture medium.
Centrifuge: Centrifuge the cell suspension to remove residual cryoprotectant and resuspend in fresh culture medium.
Culture: Plate the cells in a suitable culture vessel and incubate them under optimal conditions.
Step 8-Post-Thaw Care:
Assessment: Describe the assessment of thawed cell viability and morphology. Fill the counting chamber and recount the cells and look for any distortions.
Recovery: Allow cells to recover and grow. Monitor cell growth and function over the following days.
Complications
Ice Crystal Formation: During freezing, ice crystals can form inside the cells, and ice formation within the cells can cause mechanical injury. That is why cryoprotectants are applied to refrain from ice crystal formation.
Cryoprotectant Toxicity: DMSO (dimethyl sulfoxide) a common cryoprotectant is toxic to the cells if not washed off after thawing or if present in high concentrations.
Thawing Damage: Melting at high rates is used to prevent formation of large ice crystals, but such process, if poorly controlled can harm the cells.
Cell Viability: It is, however, noteworthy that not all the cells are capable of enduring frozen and thawed process. The cell density rate, usage of cryoprotective agents, and the type of cells are some of the aspects that determine cell viability.
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Home » Procedure » Cryopreserved Cell Suspension
Cryopreserved Cell Suspension
Updated :
August 22, 2025
Cryopreservation of cells is a cell storage procedure in which specific cells are cooled to sub-zero temperatures to be thawed subsequently for use at some other time with some level of efficacy. This technique is essential for numerous purposes, especially in the medical field, as well as disease treatment and therapeutic areas.
As the human population continues to progress into the twenty-first century, technologies of cell preservation have strategically engaged in enhancing viability of recovering the cells, protecting the cells from damage and simplifying the process.
The preservation of oocytes and embryos using vitrification, which stops the development of ice crystals by quickly chilling cells to a glass-like condition, has gained popularity.
Stem Cell Therapy: Â Cells such as hematopoietic stem cells from bone marrow or umbilical cord blood are stored and later used to treat leukemia or lymphoma.
Regenerative Medicine: The cells which are employed in regenerative medicine such as mesenchymal stem cells or adipose derived stem cells are stored for future utility.
Cell-based Vaccines: It is also possible to make some vaccines from cell lines that are either stored or lyophilized so that they can be made to release the vaccine later.
Research: Cryopreserved cell lines are used in different research activities, such as in the toxicity and efficacy of drugs, genetic disorders, and carcinogenesis.
Fertility Preservation: Cells that can be frozen include sperm, eggs, and embryos for patients who might require treatment that can affect their fertility, like chemotherapy.
Tissue Engineering: From the cell sources to generate tissue construct or organ replacement, the cells are banked frozen until they are required for implantation or other processing.
Autoimmune Conditions: When cryopreserved cells are introduced into a patient with a severe autoimmune illness, the immune response may be adversely impacted.
Allergies and Reactions: Allergies of very high severity to the cryopreservation agents or media used could be a threat.
Health Conditions: Contraindications may include severe systemic health conditions or conditions that hamper the ability of the individual’s cells to take the therapies (severe liver or kidney disease, for instance).
Cryopreservation Vials
Cryogenic Freezers
Liquid Nitrogen Storage Tanks
Cryo Boxes
Cryoprotectants
Thawing Equipment
Cryogenic Labels and Tracking Systems
Pre-Procedure Consultation: Patient assessment and making sure there are consent from the patient about their history.
Pre-Medication: Prescribe pre-medication if needed, this would be in cases where the anesthetic may cause an allergic reaction such as giving antihistamines or corticosteroids.
Thawing Cells: Thaw the cells by warming immediately the cryopreserved cell suspension in the water bath at 37°C. Avoid overheating.
Cell Washing: Gently transfer cells to a sterile centrifuge tube and wash them with an appropriate medium to remove cryoprotectants.
Patient Preparation: Ensure the patient is properly hydrated and has adequate venous access if needed for cell infusion.
Infusion: Then, procedure to add the thawed cell suspension into the patient based on the determined flow sheet, often over a period to avert adverse reactions in the patient.
Step 1-Preparation:
Cell Culture: Inoculate the flask with appropriate media to the value of the desired density. Often, cells are collected in the logarithmic growth phase to increase the chances of the harvested cells’ survival.
Equipment: Familiarize yourself with requirements about the cleanliness of the work environment like laminar flow hood and obtainable apparatus; the centrifuge, the cryovials, and the programmable freezer or cryopreservation unit.
Step 2-Harvesting Cells:
Detach Cells: For adherent cells, it is necessary to use the cell scraper or trypsin to make the cells release from the culture surface.
Wash Cells: Centrifuge the cell suspension, as this would help eliminate the culture medium and trypsin. Wash with an appropriate buffer or sequential media.
Count Cells: You should use the hemocytometer or an automated cell counter to measure the cell density.
Step 3-Prepare Cryoprotectant Solution:
Cryoprotectants: Some of the most frequently employed cryoprotective are DMSO or glycerol. Prepare the cryoprotective agent which might be DMSO (dimethyl sulfoxide) or glycerol at a concentration of 10% in either the balanced medium or serum.
Mix: Do not allow the cryoprotectant to encounter the cells before being well mixed with the cell suspension as this may cause more harm to the cells.
Step 4-Resuspend Cells:
Combine: Centrifuge & dilute the cells with the cryoprotectant solution. The general proportion is 1:1 commonly termed as a cell pellet: Cryoprotective solution ratio based on cell type and concentration.
Aliquot: Dispense the cell suspension into cryovials, usually 1-2 million cells per vial.
Step 5-Freezing:
Controlled-Rate Freezing: It is frozen at a controlled rate with a controlled rate freezer then stored at a very low temperature. This assists in avoiding the development of ice crystals that can be destructive to cells. The freezing rate is usually set at 1°C per minute at -80°C.
Step 6-Storage:
Liquid Nitrogen: After that, move the cryovials to another vial shredding container containing liquid nitrogen and store them for long term. This makes sure that the cells are well preserved at -196°C thus stopping any biological activities and retaining the functionality of the cells.
Step7-Thawing:
Warm-Up: Cryovials should be quickly thawed in a 37°C water bath with mild shaking until only a few ice crystals are visible.
Dilution: To reduce toxicity, this procedure must be done quickly, and after that, the cells should be put into the culture medium.
Centrifuge: Centrifuge the cell suspension to remove residual cryoprotectant and resuspend in fresh culture medium.
Culture: Plate the cells in a suitable culture vessel and incubate them under optimal conditions.
Step 8-Post-Thaw Care:
Assessment: Describe the assessment of thawed cell viability and morphology. Fill the counting chamber and recount the cells and look for any distortions.
Recovery: Allow cells to recover and grow. Monitor cell growth and function over the following days.
Ice Crystal Formation: During freezing, ice crystals can form inside the cells, and ice formation within the cells can cause mechanical injury. That is why cryoprotectants are applied to refrain from ice crystal formation.
Cryoprotectant Toxicity: DMSO (dimethyl sulfoxide) a common cryoprotectant is toxic to the cells if not washed off after thawing or if present in high concentrations.
Thawing Damage: Melting at high rates is used to prevent formation of large ice crystals, but such process, if poorly controlled can harm the cells.
Cell Viability: It is, however, noteworthy that not all the cells are capable of enduring frozen and thawed process. The cell density rate, usage of cryoprotective agents, and the type of cells are some of the aspects that determine cell viability.
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