Introduction

Thawing cryopreserved cells is a critical step to revive stored cells while ensuring their viability and functionality for downstream applications. This guide outlines a standardized protocol for thawing and reculturing HEK-293 cells using Pricella® Complete Medium and JET Biofil® consumables, ensuring aseptic conditions, maximum cell recovery, and reliable experimental outcomes.

Preparation

1. Biosafety Cabinet (BSC) Setup

● Place necessary tools and consumables inside the BSC before starting.

● Turn on the UV light for 30 minutes to sterilize the working environment.

● After UV exposure, disinfect the working surface with 75% ethanol to remove any remaining contaminants.

2. Water Bath Preparation

● Set the water bath to 37°C and ensure cleanliness.

● Keep a pair of disposable PE gloves nearby for safe handling of cryovials.

3. Media Pre-Warming

● Pre-warm Pricella® Complete Medium in a 37°C water bath or at room temperature.

● Transfer 9 mL of the medium into a sterile 15 mL centrifuge tube and set it aside.

Thawing Process

1. Retrieving Cryovials

● Wear protective gloves and carefully remove the cryovial from the liquid nitrogen tank.

● Avoid prolonged exposure to ambient air to prevent ice formation.

2. Rapid Thawing

● Using PE gloves, fully submerge the cryovial in the 37°C water bath.

● Gently agitate the vial to ensure uniform thawing (≤1 minute).

3. Post-Thaw Disinfection

● Wipe the cryovial surface thoroughly with 75% ethanol.

● Transfer it into the BSC for further processing.

Cell Dilution and Centrifugation

1. Transferring Cell Suspension

● Pipette the thawed cell suspension into the pre-prepared Jet Biofil® 15 mL centrifuge tube containing 9 mL of Pricella® Complete Medium.

● Mix gently to prevent shear stress.

2. Centrifugation

● Balance the tube and centrifuge at 1200 rpm (250 ×g) for 3 minutes.

3. Supernatant Removal

● Carefully discard the supernatant, retaining the cell pellet at the bottom of the tube.

Resuspension and Culturing

1. Resuspending Cells

● Add 5 mL of fresh Pricella® Complete Medium to the centrifuge tube.

● Gently pipette to resuspend the cells, avoiding bubble formation.

2. Transfer to Culture Flask

● Transfer the cell suspension into a Jet Biofil® T25 flask.

● Swirl gently for even distribution.

Quality Control

1. Microscopic Observation

● Observe cell density and morphology under a microscope.

● Capture video footage for documentation and quality assessment.

2. Incubation

● Place the T25 flask in a CO₂ incubator (37°C, 5% CO₂).

● Verify the settings for optimal growth conditions.

This protocol ensures efficient cell recovery while adhering to biosafety standards. For further inquiries, contact info@labcart.com.

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Thawing and Reculturing Cryopreserved Cells with Jet Biofil® and Pricella® HEK-293 Cell Line Revival Protocol