Cryomed controlled rate freezer

Manufactured by Thermo Fisher Scientific

Sourced in United States

The CryoMed controlled-rate freezer is a laboratory equipment used for the controlled freezing of biological samples. It provides precise temperature control and monitoring capabilities to ensure consistent and reliable freezing of materials.

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8 protocols using cryomed controlled rate freezer

Cryopreservation of Pancreatic Islet Cells

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Islet cells (1500 IEQ) were resuspended in a cryopreservation medium (90% FBS/10% DMSO) and cryopreserved using a CryoMed controlled-rate freezer (Thermo Fisher Scientific Inc., Waltham, MA, USA, Non-IVF 7452). The program used was as follows: start temperature of 0 °C, 0.2 °C/min to −40 °C, and −25 °C/min to −150 °C. Cryovials were then transferred to liquid nitrogen.
Cryopreserved islet cells were rapidly thawed in a 37 °C water bath. The thawed cells were diluted in 10 mL of culture medium and centrifuged at 280× g. After removing the supernatant, islet cells were resuspended in a standard culture medium and incubated for 24–48 h at 37 °C and 5% CO₂. Thawed islet cells were analyzed for viability using fluorescence diacetate (FDA) (0.5 µM; Sigma-Aldrich, MO, USA) and propidium iodide (PI) (75 µM; Sigma). Viability was determined by calculating the ratio of viable FDA-positive cells (green) to non-viable PI-positive cells (red). We compared the conventional protocol using an isopropanol-based freezing container (IFC) with the controlled-rate freezer (CRF) protocol. Samples in both groups (triplicate samples of the same donor for three patients) contained 1500 IEQ and were frozen in the same cryopreservation medium.

Oh J.Y., Kim Y.H., Lee S., Lee Y.N., Go H.S., Hwang D.W., Song K.B., Lee J.H., Lee W., So S., Kang E., Jun E., Shim I.K, & Kim S.C. (2022). The Outcomes and Quality of Pancreatic Islet Cells Isolated from Surgical Specimens for Research on Diabetes Mellitus. Cells, 11(15), 2335.

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Cryopreservation of Human Spermatozoa

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According to the reported studies and our results, Sperm Freezing Medium™ (ORIGIO, Målov, Denmark) was selected as the cryoprotectant for the slow freezing of spermatozoa.33 (link) The freezing medium was slowly added to the native semen sample to achieve 1:1 dilution, and the resultant mixture was packaged into 1.8 ml NuncCryotubes® (Thermo Scientific, Rockford, IL, USA). In order to reduce experimental error and guarantee quality control, a CryoMed™ Controlled-Rate Freezer (Thermo Scientific) was used for the programmed cryopreservation of spermatozoa. First, the mixture was incubated at 25°C for 5 min. Then, the temperature was decreased to −2°C at 1.2°C per min and thereafter to −45°C at 7.2°C per min. Finally, the temperature was reduced to −137°C at 25°C per min. The samples were transferred to liquid nitrogen for at least 48 h.
After storage, the samples were warmed in a 37°C water bath and shaken slightly. The postthaw sperm suspension was mixed with 5 ml G-IVF Plus medium (Vitrolife, Västra Frölunda, Sweden) and centrifuged (Centrifuge 5424 R, Eppendorf, Hamburg, Germany) at 300g for 5 min. Finally, the cell pellet was resuspended in 200 μl G-IVF Plus medium.

Zhou D., Wang X.M., Li R.X., Wang Y.Z., Chao Y.C., Liu Z.Z., Huang Z.H., Nie H.C., Zhu W.B., Tan Y.Q, & Fan L.Q. (2020). Improving native human sperm freezing protection by using a modified vitrification method. Asian Journal of Andrology, 23(1), 91-96.

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CD34+ Cell Isolation from Healthy Donors

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Leukopaks from healthy donors were purchased from HemaCare (HemaCare BioResearch Products; Van Nuys, CA). Mobilized peripheral blood (mPB) was collected from normal, healthy donors on days 5 and 6 after 5 days of stimulation with granulocyte-colony stimulating factor (G-CSF). Briefly, leukapheresis bags were washed three times with PBS/EDTA at room temperature (RT) and spun down at 150×g. Platelet depletion was performed from the centrifuged bags at each wash step using a plasma expressor extractor (Fenwal). The subsequent enrichment of CD34⁺ cells was done by using the CliniMACS Plus (Miltenyi; Bergish Gladbach, Germany). Cells were cryopreserved in CryoStor CS5 (Stemcell Technologies; Vancouver, Canada) using a CryoMed controlled-rate freezer (Thermo Fisher Scientific; Waltham, MA).

Benitez E.K., Lomova Kaufman A., Cervantes L., Clark D.N., Ayoub P.G., Senadheera S., Osborne K., Sanchez J.M., Crisostomo R.V., Wang X., Reuven N., Shaul Y., Hollis R.P., Romero Z, & Kohn D.B. (2020). Global and Local Manipulation of DNA Repair Mechanisms to Alter Site-Specific Gene Editing Outcomes in Hematopoietic Stem Cells. Frontiers in Genome Editing, 2, 601541.

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Feeder-free hiPSC Expansion and Banking

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The WTSli020 hiPSC line from fibroblasts of dermis of a healthy female that was provided by EBiSC (European Bank for induced pluripotent Stem Cells) was cultured in feeder-free conditions using Vitronectin-coated culture vessels (VTN-N; Thermo Fisher Scientific, Waltham, MA, USA) and Essential 8 Flex medium (Gibco, Grand-Island, NY, USA) supplemented with Penicillin/Streptomycin (1:1000 PenStrep; Gibco). Another hiPSC line derived from a healthy female was also used as previously described [38 (link)]. Briefly, cells were thawed and manually expanded over five supplementary passages. For manual passaging, StemPro EZPassage tool (Thermo Fisher Scientific) was used. The automated cell culture system CompacT SelecT (Sartorius, Gottingen, Germany) was then used to generate a working cell bank using 0.25 mM EDTA (Thermo Fisher Scientific) in Phosphate-Buffered Saline (PBS; Gibco) without calcium or magnesium for cell passaging. Finally, cells were dispensed into cryovials using the automated system Fill-It (Sartorius) and frozen using CryoMed Controlled-Rate Freezer (Thermo Fisher Scientific). Quality controls (mycoplasma detection, pluripotency marker expression, genomic integrity) were performed before and after amplification.

de Lamotte J.D., Polentes J., Roussange F., Lesueur L., Feurgard P., Perrier A., Nicoleau C, & Martinat C. (2021). Optogenetically controlled human functional motor endplate for testing botulinum neurotoxins. Stem Cell Research & Therapy, 12, 599.

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Cryopreservation of Bioengineered Renal Epithelial Cells

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SLA-BRECS or IM-BRECS were flushed and incubated with room temperature HTS-Purge Solution (# 637112 BioLife Solutions, Inc., Bothell, WA) for 2 minutes. HTS-Purge was then replaced with cryopreservation buffer, CryoStor 10 (CS10, #640222 BioLife Solutions) at 4°C, and the inlet and outlet were capped with sterile, Nylon, male luer integral lock ring plugs (Value Plastics, Fort Collins, CO) to prepare HREC BRECS for cryostorage. A CryoMed controlled rate freezer (Thermo Scientific, Waltham, MA) was utilized to freeze complete, individual SLA-BRECS or IM-BRECS. Briefly, this protocol consisted of a cooling rate of −1°C/min sample temperature to −4°C, followed by a rapid cooling phase during phase change, −25°C/min chamber temperature to −40°C. The chamber was then warmed 10°C/min to −14°C. After phase change, a cooling rate of −1°C/min sample temperature to −40°C was used. The last rapid cooling step was −10°C/min sample temperature to −90°C. BRECS were then directly transferred into the gas phase of a liquid nitrogen tank for cryostorage.

Pino C.J., Westover A.J., Buffington D.A, & Humes H.D. (2017). Bioengineered Renal Cell Therapy Device for Clinical Translation. ASAIO journal (American Society for Artificial Internal Organs : 1992), 63(3), 305-315.

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Cryopreservation and Thawing of Cells

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Dissociated single-cell suspensions were counted, pelleted, gently resuspended in Cryostor (BioLife Solutions, Bothell, USA), and frozen using a CryoMed™ Controlled-Rate Freezer (Thermo Scientific, Waltham, USA). After cryopreservation, vials were transferred to a liquid nitrogen tank for long-term storage. To thaw, frozen vials were placed in a 37°C water bath for 2 minutes, gently transferred to a 15 mL conical tube, and washed once with warm PBS supplemented with 0.1% BSA. Pellets were gently resuspended in either warm Dulbecco’s Modified Eagle’s Medium/Hams F-12 50/50 Mix (DMEM/F12)+Penicillin-Streptavidin (PS) or suspension culture medium.

Zook H.N., Quijano J.C., Ortiz J.A., Donohue C., Lopez K., Li W., Erdem N., Jou K., Crook C.J., Garcia I J.r., Kandeel F., Montero E, & Ku H.T. (2024). Activation of ductal progenitor-like cells from adult human pancreas requires extracellular matrix protein signaling. iScience, 27(3), 109237.

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Cryopreservation of Differentiated Cells

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To create cryopreserved cell banks for further differentiation or scRNA-seq analysis, cells were dissociated in Accutase at 37°C for 30 min on an orbital shaker, quenched with one volume of E6 medium, centrifuged, and gently resuspended in 10% dimethyl sulfoxide in E6 medium with 10 μM Y27632. The cells were aliquoted at 1 ml per cryovial and cryopreserved with a CryoMed controlled rate freezer (Thermo Fisher Scientific) using a stepwise cooling program: rapid cooling from room temperature to 4°C, 1°C/min until reaching −60°C, and 10°C/min until reaching −100°C. Cryovials were transferred to a liquid nitrogen dewer for long-term storage.

Iyer N.R., Shin J., Cuskey S., Tian Y., Nicol N.R., Doersch T.E., Seipel F., McCalla S.G., Roy S, & Ashton R.S. (2022). Modular derivation of diverse, regionally discrete human posterior CNS neurons enables discovery of transcriptomic patterns. Science Advances, 8(39), eabn7430.

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Dendritic Cell Maturation and Cryopreservation

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Monocytes were cultured in gas-permeable plastic bags (Corning, Corning, NY, USA) at a density of 5 £ 105 cells/mL in AIM-V (Gibco/ Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 800 U/mL granulocyte-macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN, USA) and 500 U/mL interleukin-4 (R&D Systems) for 5 days, followed by the addition of 10 ng/mL recombinant human tumor necrosis factor-a (R&D Systems), 5 ng/mL recombinant human interferon (IFN)-g (R&D Systems) and 2.5 mg/mL PGE2 (Cayman Chemical Company, Ann Arbor, MI, USA) into the culture bag. Mature dendritic cells were collected by centrifugation (300 x g) at day 7. For electroporation with in vitro transcribed mRNA, dendritic cells were resuspended in 4-mm gap cuvettes using the Gene Pulser X cell Electroporation System (Bio-Rad, Hercules, CA, USA) with proprietary parameters. After electroporation, cells were cultured for 4 h, collected by centrifugation, resuspended in CS10 (STEMCELL Technologies) and aliquoted in Daikyo Crystal Zenith Vials (West, Exton, PA, USA; 10 7 cells/vial). Each vial was placed in a CryoMed controlled-rate freezer (Thermo Fisher Scientific) until temperature reached to À130°C, then transferred to the liquid phase of liquid nitrogen for long-term storage until the day of distribution.

Li Q., Yang C., Tian H., Jiang J., Li P., Zhu X., Lei T., Yin R., Ding P., Bai P, & Li Q. (2023). Development of a personalized dendritic cell vaccine and single-cell RNA sequencing-guided assessment of its cell type composition. Cytotherapy, 25(2).

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