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Cryopreservation

Cryopreservation is the process of preserving living cells, tissues, or organisms at ultra-low temperatures, typically using liquid nitrogen.
This technique allows for the long-term storage and preservation of biological samples, which is essential for various medical and scientific applications, such as stem cell banking, fertility preservation, and biomedical research.
By carefully controlling the freezing and thawing processes, cryopreservation can maintain the structural and functional integrity of the preserved samples, enabling their future use.
Cryopreservation plays a crucial role in advancing medical therapies, preserving endangered species, and facilitating long-term storage of valuable biological materials.
Researchers and clinicians continually work to optimize cryopreservation protocols and techniqeus to enhance the viability and recovery of preserved samples.

Most cited protocols related to «Cryopreservation»

1
Establishment of Colorectal Cancer Xenografts
Cited 15 times
Primary CRC resection specimens were received fresh from surgery, with informed written patient consent (n = 10). All procedures were approved by the Ethics Committee of the Medical faculty, University of Rostock (Ethikkommission an der Medizinischen Fakultät der Universität Rostock, St.-Georg-Str. 108, 18055 Rostock, Germany; reference number II HV 43/2004) in accordance with generally accepted guidelines for the use of human material. Tumor samples were cut into small pieces. For cryopreservation and subsequent xenografting, pieces (3×3×3 mm) were frozen (FCS, 10% DMSO) at −80°C. Other pieces were stored in liquid nitrogen for molecular analysis. Cell culture was started from single cell suspensions, seeded on collagen-coated plates in Quantum tumor medium (+10% FCS, 2 mM L-glutamine, antibiotics and antimycotics) and incubated at 37°C in a humidified atmosphere of 5% C02. All cell culture reagents were obtained from PAA (Cölbe, Germany), antibiotics and antifungal agents were provided by the university hospital’s pharmacy.
Medium was changed regularly. Initial passage into a 25 cm2 culture flask was performed when tumor cell growth was observed. Continually growing cell cultures were further passaged and regularly stocked in low passages.
For in vivo engraftment, six-week-old female NMRI nu/nu mice were used as recipients. Mice were bred in the university’s animal facility and maintained in specified pathogen-free conditions. All experimental procedures were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Rostock (Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern; Thierfelder Str. 18, 18059 Rostock, Germany; permit number: LALLF M-V/TSD/7221.3-1.1-071-10). All surgery was performed under Ketamin/Xylazin anesthesia (dose: 90/25 mg/kg bw), and all efforts were made to minimize suffering. Subcutaneous (s.c.) tumor implantation was performed as described [13] (link). Established xenografts (≥1500 mm3) were removed and underwent in vitro culture protocols as described above.
Maletzki C., Stier S., Gruenert U., Gock M., Ostwald C., Prall F, & Linnebacher M. (2012). Establishment, Characterization and Chemosensitivity of Three Mismatch Repair Deficient Cell Lines from Sporadic and Inherited Colorectal Carcinomas. PLoS ONE, 7(12), e52485.
Publication 2012
Anesthesia Animals Animals, Laboratory Antibiotics, Antitubercular Antifungal Agents Atmosphere Cell Culture Techniques Cells Collagen Cryopreservation Ethics Committees Faculty, Medical Freezing Glutamine Heterografts Homo sapiens Magnetic Resonance Imaging Mice, Nude Mus Neoplasms Nitrogen Operative Surgical Procedures Ovum Implantation Patients Specific Pathogen Free Sulfoxide, Dimethyl Woman Xylazine
2
Establishment and Characterization of Prostate Cancer Cell Lines
Cited 14 times
LNCaP, VCaP, CWR22Rv1, PC3 and HEK293 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). All experiments with cell line were performed within 6 months of receipt from ATCC or resuscitation after cryopreservation. ATCC uses Short Tandem Repeat (STR) profiling for testing and authentication of cell lines. C4-2 and C4-2B cells were kindly provided and authenticated by Dr. Leland Chung, Cedars-Sinai Medical Center, Los Angeles, CA. The cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 0.1 mg/ml streptomycin. VCaP cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 0.1 mg/ml streptomycin. C4-2-neo and C4-2 AR-V7 cells were generated by stable transfection of C4-2 cells with either empty vector pcDNA3.1 or pcDNA3.1 encoding AR-V7 and were maintained in RPMI1640 medium containing 300 μg/mL G418. HEK293-AR-V7-PSA-E/P-LUC cells were generated by stable transfection of HEK293 cells with plasmids encoding AR-V7 and PSA-E/P-LUC reporter and were maintained in RPMI1640 medium containing 300 μg/mL G418. C4-2B cells were chronically exposed to increasing concentrations of enzalutamide (5 ~ 40 μM) by passage in media containing enzalutamide for >12 months in complete FBS and stored for further analysis. Cells resistant to enzalutamide were referred to as C4-2B MR (C4-2B enzalutamide resistance)(18 (link)). Parental C4-2B cells were passaged alongside the enzalutamide treated cells as an appropriate control. C4-2B MR cells were maintained in 20 μM enzalutamide containing medium. All cells were maintained at 37°C in a humidified incubator with 5% carbon dioxide.
Liu C., Lou W., Zhu Y., Nadiminty N., Schwartz C.T., Evans C.P, & Gao A.C. (2014). Niclosamide inhibits androgen receptor variants expression and overcomes enzalutamide resistance in castration resistant prostate cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 20(12), 3198-3210.
Publication 2014
antibiotic G 418 Carbon dioxide Cell Line Authentication Cell Lines Cells CHOP regimen Cloning Vectors Cryopreservation enzalutamide Fetal Bovine Serum HEK293 Cells Parent Penicillins Plasmids Resuscitation Short Tandem Repeat Streptomycin Transfection
3
Comprehensive Pancreas Tissue Collection
Cited 13 times
Pancreas recovery was performed as for a standard transplantation and included intra-operative flushing in the majority of cases. The pancreas with fat containing pancreatic lymph nodes and proximal duodenum (stapled closed) were placed in a sterile container with additional media, submerged in ice, and shipped by standard organ courier to the University of Florida. Spleen and non-pancreatic lymph nodes were also recovered as were serum and whole blood tubes and shipped with the pancreas. The donor processing team dissected tissues and prepared a variety of storage formats for each sample type. The pancreas was weighed then divided into regions (head, body, tail). Each region was transected into alternating sections for fixed (formalin) and fresh frozen blocks. Minced samples were snap frozen in liquid nitrogen with or without RNAse inhibitor (RNAlater, Ambion, CA). Spleen and lymph node samples were processed in two main formats, the first as for the pancreas and the second for cryopreserved cells. In addition, fresh spleen and pancreatic lymph nodes were also minced and placed into sterile media for immediate shipment to investigators. Whole blood was processed to peripheral blood mononuclear cells (PBMC) using Ficoll density gradient followed by cryopreservation (13 (link)). All samples and aliquots were inventoried in an online database with reservation of approximately 30% of each sample type to ensure availability for next generation analytics.
Campbell-Thompson M., Wasserfall C., Kaddis J., Albanese-O’Neill A., Staeva T., Nierras C., Moraski J., Rowe P., Gianani R., Eisenbarth G., Crawford J., Schatz D., Pugliese A, & Atkinson M. (2012). Network for Pancreatic Organ Donors with Diabetes (nPOD): Developing a Tissue Biobank for Type 1 Diabetes. Diabetes/metabolism research and reviews, 28(7), 608-617.
Publication 2012
BLOOD Cryopreservation Duodenum Ficoll Formalin Freezing Head Human Body Nitrogen Nodes, Lymph Pancreas PBMC Peripheral Blood Mononuclear Cells Ribonucleases Serum Spleen Sterility, Reproductive Tail Tissue Donors Transplantation
4
Malaria Parasite Isolation and Purification
Cited 13 times
Clinical isolates of P. vivax and P. falciparum were collected from malaria patients receiving treatment from clinics run by the Shoklo Malaria Research Unit on the North Western border of Thailand. All patients were briefed on the project and provided informed consent prior to collection of blood by venipuncture. Five milliliters of whole blood was collected in lithium heparin collection tubes. This sample was either cryopreserved directly in Glycerolyte 57 (Baxter) or leukocyte depleted using CF11 cellulose columns 38 (link) prior to cryopreservation.
P. falciparum (3D7 clone of the NF54 strain) samples were obtained from cultures performed using sealable flasks using RPMI-HEPES medium at pH 7.4 supplemented with hypoxanthine 50 μg/mL, NaHCO3 25 mM, gentamicin 2.5 μg/mL, and Albumax II (Gibco, Singapore) 0.5% wt/vol in an atmosphere containing 5% CO2, as previously described 39 (link).
The selection of the late developmental stages of P. falciparum containing the hemozoin pigment was performed using the MACS system (Miltenyi, Singapore). One to two milliliters of blood at 50% hematocrit in PBS were passed through the LD column as described elsewhere 40 (link). After washing twice with 2 ml of PBS, the late developmental stages were collected from the magnetically-retained fraction.
Malleret B., Claser C., Ong A.S., Suwanarusk R., Sriprawat K., Howland S.W., Russell B., Nosten F, & Rénia L. (2011). A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development. Scientific Reports, 1, 118.
Publication 2011
Atmosphere Bicarbonate, Sodium Blood Cellulose Clone Cells Cryopreservation Gentamicin hemozoin Heparin HEPES Hypoxanthine Leukocytes Lithium Malaria Patients Phlebotomy Pigmentation Strains Volumes, Packed Erythrocyte
5
Isolation and Culture of Adipose-Derived Cells
Cited 12 times
Adipose tissue was transferred to the lab in medium 199 at room temperature. After mincing into approximately 5 to 10 mg fragments, tissue was digested with collagenase (type 1, 1 mg/ml in HBSS) for 2 hours at 37ºC with shaking (100 rpm) (9 (link)). The mixture was passed through a 250 micron mesh, centrifuged at 500g for 10 minutes and floating adipocytes were discarded. Cell pellets were treated with erythrocyte lysis buffer (0.154mM NH4Cl, 10 mM K2HPO4 and 0.1 mM EDTA, pH 7.3) for 10 min at room temperature. After discarding the supernatant, cells were repelleted with centrifugation, resuspended in growth media (α-MEM with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin) and then plated. On the following day, cells were washed with PBS and replenished with growth media. At 70~80% confluence, cells were either subcultured or frozen (10% DMSO supplemented growth media) for cryopreservation.
Lee M.J., Wu Y, & Fried S.K. (2012). A modified protocol to maximize differentiation of human preadipocytes and improve metabolic phenotypes. Obesity (Silver Spring, Md.), 20(12), 2334-2340.
Publication 2012
Adipocytes Buffers Cells Centrifugation Collagenase Cryopreservation Culture Media Edetic Acid Erythrocytes Freezing Hemoglobin, Sickle Pellets, Drug Penicillins potassium phosphate, dibasic Streptomycin Sulfoxide, Dimethyl Tissue, Adipose Tissues

Most recents protocols related to «Cryopreservation»

1
Cell Bank Thawing and Characterization
Not available on PMC !

Example 4

This example demonstrates thawing of cell bank samples for use in characterization assays and for products.

The frozen cryopreservation vials are placed in a heat block set at 37° C.±1.5° C. for approximately 5 minutes.

Once thawed, the contents are transferred to a new tube. The cryopreservation vial is rinsed with EG2MX solution and transferred to the new tube. The volume is increased to 10 mL with EG2MX.

Cells are counted for viability using a hemacytometer. Using a microscope, the middle square and each corner square are counted. Viable and non-viable cells are counted and the number of viable cells/mL is calculated. The percent of cells recovered after thaw is calculated. The percent recovery should be at least 50%.

To initiate cell culture, 35 mL of EG2MX solution is inoculated with 5 mL of cell suspension in a flask. Cells are grown in a 5% CO2 incubator at 37° C.±1.5° C.

US11857576B2. Compositions and methods for repairing cartilage defects (2024-01-02). Vericel Corporation [US]. Inventors: Ross A. Tubo [US], Xing Zhao [US].
Patent 2024
Biological Assay Cell Culture Techniques Cells Cryopreservation Freezing Microscopy
2
Vitreous Sample Collection and RNA Extraction
A blood sample (approximately 6 mL) was drawn from the forearm vein into a tube containing an anticoagulant following overnight fasting on the day of surgery. The mixture was immediately centrifuged at 4000×g for 10 min at 4 °C. A vitreous sample (approximately 1 mL) was carefully collected into a 2 mL sterile syringe using a 25-gauge vitreous cutter and manual suction before opening the intraocular irrigation system. If vitreous hemorrhage was present, the surgeon avoided collecting blood components as much blood as possible. All samples were stored in cryopreservation tubes and immediately cooled at − 80 °C until analysis.
After sample collection, total RNA was extracted using TRIzol LS reagent (Invitrogen, Carlsbad, CA, USA) combined with miRNeasy Micro Kit (Qiagen, Hilden, Germany). RNA quality and integrity were measured using Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA) and Agilent 4200 TapeStation.
Zeng L., Zhou M., Wang X., Long X., Ye M., Yuan Y, & Tan W. (2023). Aberrant lncRNA expression in patients with proliferative diabetic retinopathy: preliminary results from a single-center observational study. BMC Ophthalmology, 23, 94.
Publication 2023
Anticoagulants BLOOD Blood Component Transfusion Cryopreservation Forearm Specimen Collection Sterility, Reproductive Suction Drainage Surgeons Surgery, Day Syringes trizol Veins Vitreous Hemorrhage
3
Ovarian Stimulation Protocols: A Comparative Evaluation
Ovarian stimulation protocols included gonadotropin-releasing hormone (GnRH) agonist protocol, GnRH antagonist protocol, and progestin-primed ovarian stimulation (PPOS) protocol. Recombinant human chorionic gonadotropin (OVIDREL; Merck Serono, Darmstadt, Germany) or GnRH-a (Decapeptyl; Ferring, Saint-Prex, Switzerland) were administered in patients when two leading follicles reached 18 mm in diameter. Oocyte retrieval was performed at 36 h after recombinant human chorionic gonadotropin or GnRH-a triggered by transvaginal ultrasound-guided aspiration. Insemination method was selected according to the sperm count after sperm preparation. A morphologic score of cleavage-stage embryo was given based on the number of blastomeres, the homogeneous degree of blastomeres, and the degree of cytoplasmic fragmentation, which has been extensively described in our previous study (3 (link)). If a couple has two or more high-quality cleavage-stage embryos on day 3 of embryo culture, the embryos were selected and cultured to blastocyst stage. Blastocyst evaluation was performed according to the Gardner’s grading system (4 (link)).
For patients who underwent GnRH agonist protocol and GnRH antagonist protocol, one to two fresh embryos were transferred into the uterus of women free of OHSS, hydrosalpinx, intrauterine adhesion and high progesterone level (> 1.5 ng/ml) on the day of triggering, and then, the spare embryos were cryopreserved for the next FET. Patients who underwent PPOS protocol had to freeze all their embryos. The vitrified cryopreservation was conducted according to standard protocols, as previously described (5 (link)).
Fan L., Li N., Liu X., Li X., Cai H., Pan D., Wang T., Shi W., Qu P, & Shi J. (2023). Hormone replacement treatment regimen is associated with a higher risk of hypertensive disorders of pregnancy in women undergoing frozen-thawed embryo transfer. Frontiers in Endocrinology, 14, 1133978.
Publication 2023
Blastocyst Blastomeres Cleavage Stage, Ovum Cryopreservation Cytoplasm Decapeptyl Embryo Freezing Gonadorelin Hair Follicle Human Chorionic Gonadotropin Insemination Oocyte Retrieval Ovarian Hyperstimulation Syndrome Ovarian Stimulation Ovidrel Patients PRIME protocol Progesterone Progestins Sperm Ultrasonography Uterus Woman
4
Blood Sample Processing and Storage
Whole blood and serum samples were obtained from volunteers at each visit to the Clinical Research Center of Tel Aviv Sourasky Medical Center and transferred within 2 hours of collection to the School of Public Health Research Lab, at the Sackler Faculty of Medicine, Tel Aviv University for further processing and storage. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood followed by activation or cryopreservation. Serum samples were centrifuged within 4 hours after collection and stored at -80 °C until further analyzed. The assay procedures and analyses of all parameters described below were identical in the phase I and follow-up studies. Samples of volunteers collected in the phase I study were retested in the same runs with the new samples obtained during the follow up. The results obtained at retesting were in all cases below 2-fold change in titer compared to the original data.
Meron-Sudai S., Asato V., Adler A., Bialik A., Goren S., Ariel-Cohen O., Reizis A., Mulard L.A., Phalipon A, & Cohen D. (2023). A Shigella flexneri 2a synthetic glycan-based vaccine induces a long-lasting immune response in adults. NPJ Vaccines, 8, 35.
Publication 2023
Biological Assay BLOOD Cryopreservation Faculty, Medical PBMC Peripheral Blood Mononuclear Cells Serum Voluntary Workers
5
Assessing Apple Storability and Nutrient Retention
Immediately after harvest, the apples were transported to refrigerated air storage where they were held for 150 d at 1°C and RH of 90–95%. After removing fruit from cold storage, they were kept for 7 d at 20°C and 60–70% RH to simulate the shelf life period.
Apple storability was assessed based on FF, SSC, TA, the weight loss of fruit during the storage and shelf life periods, and the incidence of physiological disorders and fungal diseases.
Firmness, SSC and TA of fruit were determined on 30 apples from each plot according to the same procedures as at harvest.
Fruit disorders and diseases were visually evaluated after the shelf life period on a 20 kg bulk sample from each plot. To assess disorders/diseases developed in the inner cortex, each fruit was cut longitudinally into four parts. The number of fruit affected by each disorder or disease was recorded. The results were expressed as a percentage share of affected fruit compared to the total number of fruit in the bulk sample.
Since Ca ions do not tend to volatilise, only the loss rates of I and Se from stored apples were determined. The losses were calculated by subtracting from amounts I or Se in edible parts of fruit at harvest and their amounts after the shelf life period. Fruit I and Se after storage were determined on 35 apples from the plot in the same manner as at harvest.
, & Wójcik P. (2023). Effects of preharvest sprays of iodine, selenium and calcium on apple biofortification and their quality and storability. PLOS ONE, 18(3), e0282873.
Publication 2023
ARID1A protein, human Cortex, Cerebral Cryopreservation Dietary Fiber Fruit Ions Mycoses physiology

Top products related to «Cryopreservation»

1
Dimethyl sulfoxide (dmso) by Merck Group
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DMSO is a versatile organic solvent commonly used in laboratory settings. It has a high boiling point, low viscosity, and the ability to dissolve a wide range of polar and non-polar compounds. DMSO's core function is as a solvent, allowing for the effective dissolution and handling of various chemical substances during research and experimentation.
2
Fetal bovine serum (fbs) by Thermo Fisher Scientific
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Fetal Bovine Serum (FBS) is a cell culture supplement derived from the blood of bovine fetuses. FBS provides a source of proteins, growth factors, and other components that support the growth and maintenance of various cell types in in vitro cell culture applications.
3
Streptomycin by Thermo Fisher Scientific
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Streptomycin is a broad-spectrum antibiotic used in laboratory settings. It functions as a protein synthesis inhibitor, targeting the 30S subunit of bacterial ribosomes, which plays a crucial role in the translation of genetic information into proteins. Streptomycin is commonly used in microbiological research and applications that require selective inhibition of bacterial growth.
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Penicillin is a type of antibiotic used in laboratory settings. It is a broad-spectrum antimicrobial agent effective against a variety of bacteria. Penicillin functions by disrupting the bacterial cell wall, leading to cell death.
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DMEM (Dulbecco's Modified Eagle's Medium) is a cell culture medium formulated to support the growth and maintenance of a variety of cell types, including mammalian cells. It provides essential nutrients, amino acids, vitamins, and other components necessary for cell proliferation and survival in an in vitro environment.
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Penicillin streptomycin by Thermo Fisher Scientific
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Penicillin/streptomycin is a commonly used antibiotic solution for cell culture applications. It contains a combination of penicillin and streptomycin, which are broad-spectrum antibiotics that inhibit the growth of both Gram-positive and Gram-negative bacteria.
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Ficoll-Paque PLUS is a sterile, ready-to-use medium for the isolation of mononuclear cells from blood or bone marrow by density gradient centrifugation. It is a polysucrose and sodium diatrizoate solution with a density of 1.077 g/mL.
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L glutamine by Thermo Fisher Scientific
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L-glutamine is an amino acid that is commonly used as a dietary supplement and in cell culture media. It serves as a source of nitrogen and supports cellular growth and metabolism.
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GlutaMAX is a chemically defined, L-glutamine substitute for cell culture media. It is a stable source of L-glutamine that does not degrade over time like L-glutamine. GlutaMAX helps maintain consistent cell growth and performance in cell culture applications.

More about "Cryopreservation"

Cryopreservation is the process of preserving living cells, tissues, or organisms at ultra-low temperatures, typically using liquid nitrogen.
This technique allows for the long-term storage and preservation of biological samples, which is essential for various medical and scientific applications, such as stem cell banking, fertility preservation, and biomedical research.
By carefully controlling the freezing and thawing processes, cryopreservation can maintain the structural and functional integirty of the preserved samples, enabling their future use.
Cryopreservation plays a crucial role in advancing medical therapies, preserving endangered species, and facilitating long-term storage of valuable biological materials.
Researchers and clinicians continually work to optimize cryopreservation protocols and techniques to enhance the viability and recovery of preserved samples.
DMSO (dimethyl sulfoxide) is a commonly used cryoprotectant that helps protect cells during freezing, while FBS (fetal bovine serum) and supplements like streptomycin, penicillin, DMEM (Dulbecco's Modified Eagle Medium), and L-glutamine are often used in cryopreservation media to support cell health and survival.
Additionally, Ficoll-Paque PLUS and Lymphoprep are density gradient media used to isolate and purify specific cell types for cryopreservation.
Optimizing cryopreservation protocols and techniques is essential for advancing medical therapies, preserving endangered species, and facilitating long-term storage of valuable biological materials.
Researchers and clinicians continue to explore new methods and strategies to improve the viability and recovery of cryopreserved samples, driving progress in fields like regenerative medicine, fertility preservation, and biomedical research.