Ask A Scientist: How Does Astarte Cryopreserve Cells?

As a buyer of human and animal immune cell products, you trust that your biomaterials will be handled with care and precision throughout the collection, processing, characterization, and cryopreservation processes. We understand how immensely important cell quality is to the efficacy and timeliness of your research efforts, which is why we are completely transparent with our cryopreservation method.

We are often asked how we cryopreserve our cells, either by curious researchers looking for the best protocol or by scientists validating our quality processes. We have experimented with many cryopreservation methods over the years to find the best protocol that ensures a viable product.

Careful cryopreservation of cells affords you greater flexibility in planning experiments, controls for certain variables, and ensures you can make full use of your precious human biomaterials. The advantages of cryopreserved cells far outweigh the potential downside in most situations.

In-depth: Explore our case study on the timing of isolation and cryopreservation and the impact on cell viability and functionality.

Astarte’s Proven Cryopreservation Methods & Tips

Select the Right Medium

  • We prefer a simple cryopreservation medium that can be prepared with clinical-grade materials. We use phosphate-buffered saline, 2 g/100mL human albumin 10% DMSO.
  • This concentration of albumin is within the range typically found in serum, which allows us to mimic the serum environment without the risk of lot-to-lot variation when using actual serum.
  • We prepare the cryopreservation medium as  two components: one that is just PBS with 2% albumin that is used for preparing a well dispersed cell suspension ( so the cells can be well-dispersed in a 2% albumin solution with no DMSO). The second component is PBS 2% albumin and 20% DMSO.  This “FreezeB” solution is added to the cell suspension at a 1:1 ratio to achieve a final concentration of 10% DMSO. This method avoids the need to resuspend in the presence of DMSO, which risks damaging the cell membranes. 

Cryopreserve Quickly

  • We cryopreserve our cells as soon after collection as possible to preserve freshness and viability, typically within 8 hours. 
  • Cells that are cryopreserved more than 24 hours after collection can still have high viability prior to freezing, but remember that viability dips slightly after thaw, so time is of the essence. 
Post-thaw viability of cells isolated and cryopreserved the same day as collection.

Aliquot Rapidly

  • We see a noticeable decrease in viability when preparing large batches for cryopreservation. To avoid this, try not to aliquot more than 50 vials at a time to ensure each batch gets in the freezer quickly. 

Control the Freeze Rate

  • Freezing your biomaterials too quickly can cause the formation of ice crystals, which can damage the cells. The recommended freeze rate is 1°C per minute. 
  • Achieve a slower freeze rate by placing vials in an insulated container. We use either simple foam boxes or Mr. Frosty isopropyl alcohol freezing containers for insulation.
  • You can purchase a controlled rate freezer to achieve the same result, which is best when freezing cells in bags rather than vials.  

Rapidly Thaw

  • Rapid thawing is important with immune cells, but be careful not to overheat the sample. Follow our proven thawing protocol:
    • Place cells in a 37°C water bath
    • Agitate until completely thawed
    • Transfer thawed cells to warm medium as soon as the ice melts; dilute the DMSO at least ten-fold
    • Mix gently by inversion or pipetting
    • Centrifuge for 10 minutes at 200 x g
    • Aspirate or decant the supernatant and resuspend the cell pellet
    • Remove an aliquot for cell count and proceed with the experiment

Keep this infographic handy at your bench, or share with a colleague. A simple and effective cell thawing protocol. Get it here: Click To Tweet


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How to Thaw Cells

Materials

  • Frozen Cells
  • 37°C Water Bath
  • Culture Medium with 5-10% Serum
  • Centrifuge

Procedure

  1. Place Cells in a 37°C water bath and agitate until thawed
  2. Add Thawed Cells to 9mL of warm serum-containing medium
  3. Mix Gently by inverting tube 2–3 times or pipetting up and down several times
  4. Centrifuge for 10 minutes at 200 x g
  5. Aspirate or Decant the supernatant
  6. Resuspend the cell pellet in 10mL of medium

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