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Serum-free cell cryopreservation solution
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- Description
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Product Background Overview
Cell cryopreservation media serve as the liquid environment during cryopreservation, providing nutrients and protective effects to cells. They lower the freezing point, increase the permeability of cell membranes to water, and facilitate the efflux of intracellular water prior to freezing, thereby preventing or minimizing ice-crystal-induced damage. This allows cells to be temporarily suspended from active growth while preserving their intrinsic characteristics, so that they can be directly revived when needed to regain viability. Traditionally, cell cryopreservation media are prepared by mixing culture medium, serum, and DMSO in specific ratios; however, due to the complex composition of serum and significant batch-to-batch variability, their application is limited, and they require a stepwise cooling protocol for cryopreservation, which is time-consuming and labor-intensive.
HeYouSheng’s newly launched serum-free cell cryopreservation solution is primarily intended for NK cells and γδ T cells T cells, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), embryonic stem (ES) and induced pluripotent stem (iPS) cells, mesenchymal stem cells (MSCs), and other cell types have been validated to exhibit superior performance compared with comparable imported products.
1. Serum-free and free of animal-derived components.
2. Store at 2–8°C; no need for repeated freeze-thaw cycles.
[Product Principle]
Cell cryopreservation solutions contain a variety of cryoprotectants: some readily penetrate cells to prevent intracellular water from forming ice crystals that could damage the cells, while others cannot penetrate cells but can preferentially bind extracellular water molecules before ice crystal formation, thereby reducing the electrolyte concentration in the extracellular solution and limiting the influx of cations into the cell. Our company’s serum-free cell cryopreservation solution is formulated as a combination of multiple cryoprotectants, providing dual protection both inside and outside the cell and significantly enhancing cell recovery viability. The composition of our cell cryopreservation solution is well-defined, making it safer and more effective.
【Product Advantages】
Safety: Serum-free, xenogeneic-component-free;
Precise positioning: It is suitable for the storage of immune cells and stem cells, with particularly excellent cryopreservation results for NK cells and γδ T cells.
Efficiency: High cell recovery rates, with recovery viability exceeding 90% for multiple cell types;
Convenience: Ready-to-use; no additional preparation required.
Simplicity: Suitable for cryopreservation of immune cells and stem cells, as well as long-term storage in liquid nitrogen at −196°C; simplified protocols for additional cell lines require validation before use.
Product Usage Instructions
1. Preparations Before Cell Cryopreservation
1) The viability of cryopreserved cells must be greater than 90% (the buffy coat fraction obtained from peripheral blood can be cryopreserved immediately).
2) Calculate cell density; refer to the attached table for recommended cryopreservation densities for different cell types.
2. Cell Cryopreservation Process
1) Count the cell suspension to be cryopreserved, then centrifuge at 400 × g for 10 minutes.
2) Discard the supernatant, then slowly add the 4°C–stored cell cryopreservation solution to the pelleted cells, adjusting the volume of the cryopreservation solution based on cell density.
3) After repeated pipetting and mixing, aliquot the solution into cryovials, 500–1000 µL per vial.
4) Place the aliquoted cell cryovials in a programmed cooling box or a programmable freezer, then transfer them to liquid nitrogen for long-term storage.
Note: Cell cryovials must be completely sealed; otherwise, they may rupture during the thawing process.
3. Cell Revival Process
1) Turn on the water bath in advance and set the temperature to 37°C.
2) Remove the cryovial from liquid nitrogen and promptly place it in a water bath to thaw, aiming to complete thawing within 1 minute; the shorter the thawing time, the lesser the impact on the cells.
3) Quickly dispense the melted cell-containing cryopreservation solution into fresh culture medium and mix thoroughly. For example, if the cryopreservation solution volume is 500 µL, add it to 5 mL of complete culture medium; if the cryopreservation solution volume is 1 mL, add it to 10 mL of complete culture medium.
4) After thorough mixing, centrifuge immediately at 400 × g for 10 minutes. Upon completion of centrifugation, discard the supernatant and add pre-warmed fresh culture medium.
5) Slowly add an appropriate volume of fresh culture medium to the cell pellet, gently mix to ensure uniform suspension, and then transfer the well-mixed cells into pre-prepared culture vessels. After mixing, aliquot the cells into flasks and incubate in a CO2 incubator (with the CO2 concentration determined according to the requirements of the culture medium, typically 5%).
Appendix:
Precautions:
1. During cryopreservation, after adding the cryoprotective solution to the cells, perform all procedures as close as possible to an ice pack, as low temperatures help prevent damage to the cells caused by the cryoprotectant.
2. Ensure that cryovials are completely sealed to prevent rupture during the thawing process.
3. This product is supplied in sterile packaging and does not require filtration; please ensure aseptic handling during use.
4. For your safety and health, please wear a lab coat and disposable gloves during operation.
Presentation of Application Data for Serum-Free Cell Cryopreservation Solution
1. Viability of Cryopreserved PBMCs
Cryopreservation of PBMCs, followed by recovery and assessment of cell count and viability, with a comparative chart against the three leading serum-free cryopreservation media on the market.
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All maintained relatively consistent post-resuscitation cell counts.
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Cell viability consistently exceeds 95%.
2. Cryopreservation and sorting of PBMC monocytes
PBMCs cryopreserved in four different cryoprotective solutions were thawed and subjected to magnetic bead-based sorting for monocytes, with the purity and viability of the sorted monocytes subsequently compared.
Conclusion: Cell viability after sorting was greater than 90% for both Hycells cryopreservation medium and Cryopreservation Medium No. 4; moreover, the purity of mononuclear cells following sorting after cryopreservation of PBMCs was significantly higher when Hycells cryopreservation medium was used compared with other cryopreservation media.
3. Sorting monocytes to induce macrophages and their phagocytic activity
1) Compare the induction of macrophages using cryopreservation solution No. 4, which exhibits relatively good single-nucleus sorting performance, with Hycells cryopreservation solution; microscopic observation:
Conclusion: Microscopic examination on day 5 of induction revealed that cells cryopreserved in Hycells cryopreservation medium exhibited prominent dendritic differentiation, with a higher induction success rate compared with cryopreservation medium No. 4.
2) Compare the induction of macrophages using single-nucleus sorting–optimized cryopreservation solution No. 4 with Hycells cryopreservation solution, and assess the expression of macrophage-associated markers.
Conclusion: Hycells cryopreservation solution demonstrates significantly superior performance in the expression of macrophage markers such as CD24, CD80, CD83, and CD206 compared with Cryopreservation Solution No. 4.
Partial streaming raw data:
3) Induce macrophages using cryopreservation solutions from competitors No. 1, No. 3, and No. 4, and compare their performance with that of Hycells’ No. 2 cryopreservation solution, then assess phagocytosis.
Conclusion: Both Hycells cryopreservation solution and Cryopreservation Solution No. 3 exhibited significant phagocytic activity in macrophages across three batches, with relatively consistent performance across batches.
4. GDT cell expansion following thawing and recovery of cryopreserved PBMCs
1) Cells from different batches, cryopreserved separately using Cryo-4 and Hycell cryopreservation solution, were expanded using HeYouSheng’s proprietary GDT expansion kit. (Item No.: GD990-1L) Perform GDT amplification and observe cellular proliferation under the microscope.
Conclusion: Based on the microscopic observations on Day 3 and Day 11, Hycells cryopreservation medium consistently demonstrated significantly superior cell clumping and growth compared with Cryopreservation Medium No. 4 across both batches.
Amplification factor
Purity data
Conclusion: In terms of expansion fold, all three batches of cells cryopreserved in Hycells cryopreservation solution exhibited varying degrees of expansion, whereas none of the three batches of cells cryopreserved in a comparable imported product showed significant expansion.
Based on the expansion purity data, the GDT cells expanded in Hycells cryopreservation medium exhibited a steady increase in purity over the course of expansion, with all three donor samples achieving a purity of greater than 90% by day 14.
Conclusion: Following cryopreservation and thawing with Hycells cryopreservation solution, the cytotoxicity of GDT cells did not decrease significantly, and the cytotoxicity of Hycells cryopreservation solution was consistently superior to that of a comparable imported cryopreservation solution.
5. Summary of Viability and Overnight Data for Cryopreserved, Expanded NK/GDT Cells
1) Two donors’ expanded NK and GDT cells were cryopreserved and subsequently thawed; the cells were then cultured overnight for 24 hours, after which cell viability was assessed.
GDT cells
NK cells
Conclusion: After thawing and culturing for 24 hours, the viability of expanded NK/GDT cells cryopreserved in Hycells Cryopreservation Solution No. 2 shows no significant decline.
When NK/GDT cells expanded and then cryopreserved using the No. 4 equivalent imported cryopreservation solution, the post-thaw viability decreased more markedly after overnight recovery.
For more details on HeYouSheng Bio’s serum-free cell cryopreservation solution, please contact our sales representative. Scan the QR code below to request a free trial sample.
Contact: Mr. Xie 15201775322 。
Download related documents for serum-free cell cryopreservation solution:
Serum-Free Cell Cryopreservation Solution – 20 mL Datasheet
Serum-Free Cell Cryopreservation Solution – 100 mL Data Sheet
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Related document downloads:
Serum-Free Cell Cryopreservation Solution – 100 mL Data Sheet
Serum-Free Cell Cryopreservation Solution – 20 mL Datasheet
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