Human primary cells are cells isolated directly from tissues, including peripheral blood, cord blood, and bone marrow. These cells are increasingly recognized for their importance in the study of biological processes, disease progression, and drug development, and for applications including in vitro cell-based assays or the creation of xenograft or humanized mouse models.

Fresh primary cells are often cryopreserved (i.e. frozen) for long-term storage in liquid nitrogen if they are not required immediately. Researchers with limited resources for processing fresh samples may also purchase frozen, ready-to-use primary cells, including mononuclear cells (MNCs), purified immune cells, or hematopoietic stem cells. When thawing frozen cells, proper technique and handling ensures optimal viability, recovery, and functionality of the cells for downstream applications.

This protocol describes how to thaw frozen primary cells. As thawing protocols for specific cell types may vary, always refer to the recommended protocol received with your cells.

Materials

• Human primary cells (frozen)
• ThawSTAR® CFT2 Automated Thawing System (Catalog #100-0650)
• Recommended medium. Options include:
  • Iscove's Modified Dulbecco's Medium (IMDM, Catalog #36150) with 10% Fetal Bovine Serum (FBS; Catalog #100-0179) added
  • DMEM with 4500 mg/L D-Glucose (Catalog #36250) with 10% Fetal Bovine Serum (FBS; Catalog #100-0179) added
  • RPMI 1640 Medium (Catalog #36750) with 10% Fetal Bovine Serum (FBS; Catalog #100-0179) added
  • Phosphate-buffered saline with 2% FBS (e.g. Dulbecco's Phosphate Buffered Saline with 2% Fetal Bovine Serum, Catalog #07905)
• 2 mL serological pipettes (e.g. Falcon® Serological Pipettes, 2 mL, Catalog #38002)
• 25 mL serological pipettes (e.g. Falcon® Serological Pipettes, 25 mL, Catalog #38005)
• 50 mL conical tubes (e.g. Falcon® Conical Tubes, 50 mL, Catalog #38010)
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• DNase I Solution (1 mg/mL) (Catalog # 07900)
• Hausser Scientific™ Bright-Line Hemocytometer (Catalog #100-1181)
• Trypan Blue (Catalog #07050)
• Pipettor (e.g. Corning® Lambda™ Plus Pipettor, Catalog #38060)
• Pipette tips (e.g. Corning® Filtered Pipette Tips, Catalog #38034)
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Protocol

Part I: Setup

1. Warm medium in a 37°C water bath. See Materials for list of recommended media. If thawing cells for downstream cell separation, PBS with 2% FBS can be used.
2. When removing frozen cells from storage, it is important to minimize exposure to room temperature (15 - 25°C). If not proceeding directly to thawing, place the cells on dry ice or in a liquid nitrogen container.



Part II: Thawing Cells

Note: It is recommended to thaw only one frozen cell vial at a time to prevent prolonged exposure to DMSO at higher temperatures.
3. Wipe the outside of the vial of cells with 70% ethanol or isopropanol.
4. In a biosafety cabinet, twist the cap a quarter-turn to relieve internal pressure and then retighten.
5. Quickly thaw cells in a 37°C water bath by gently swirling the vial. Remove the vial when a small amount of ice remains. This should take approximately 1 - 2 minutes. Do not vortex the cells. Alternatively, use the ThawSTAR® CFT2 Automated Thawing System for ensured sample sterility and consistent thawing performance. For more information refer to the following video: How to Quickly Thaw Frozen Cells with the ThawSTAR® CFT2 Automated Thawing System.



Part III: Cell Washing and Counting

Note: It is important to work quickly in the following steps to ensure high cell viability and recovery.
6. Wipe the outside of the vial again with 70% ethanol or isopropanol.
7. In a biosafety cabinet, measure the total volume of the cell suspension using a 2 mL serological pipette. This value is used in step 13 to calculate the total number of cells provided. Place the cells back into the vial to mix the suspension.
8. Remove a 20 µL aliquot of cells for counting. If using Trypan Blue to assess viability, for ≥ 1 x 10⁶ cells add a minimum of 20 µL of medium and record the volume of medium added. For < 1 x 10⁶ cells, dilute directly in 20 µL of Trypan Blue. Set diluted aliquot aside until step 13. For more details on performing cell counts with a hemocytometer, please refer to the following Protocol: How to Perform Cell Counts with a Hemocytometer.
   
   
   Important: A viable cell count must be done on an aliquot collected immediately after thawing (before washing). This will confirm the number of cells provided, and track potential cell loss in the wash process. Cell loss of up to 30% can be expected during the wash steps.
9. Transfer the remaining cell suspension to a 50 mL conical tube using a pipette.
10. Rinse the vial with 1 mL of medium and add it dropwise to the cells, while gently swirling the 50 mL tube.
11. Wash by adding 15 - 20 mL of medium dropwise, while gently swirling the tube.
12. Centrifuge the cell suspension at 300 x g for 10 minutes at room temperature (15 - 25°C).
13. If using Trypan Blue, perform a cell count on the diluted aliquot from step 8.
14. Carefully remove the supernatant (from step 12) with a pipette, leaving a small amount of medium to ensure the cell pellet is not disturbed. Resuspend the cell pellet by gently flicking the tube.
15. If cells are starting to clump, add 100 µg DNase I Solution per mL of cell suspension and incubate at room temperature for 15 minutes.
    
    
    Note: Do not add DNase I Solution if the cells will be used for DNA or RNA extraction.
16. Gently add 15 - 20 mL of medium to the tube.
17. Centrifuge the cell suspension at 300 x g for 10 minutes at room temperature.
18. Carefully remove the supernatant with a pipette, leaving a small amount of medium to ensure the cell pellet is not disturbed. Resuspend the cell pellet by gently flicking the tube.
19. Cells are now ready for use in downstream applications, such as cell culture with MethoCult™ or ImmunoCult™, and cell isolation with EasySep™.