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Shanghai Jinma Bio has developed a practical and reliable method for preserving and transporting cells commonly used by our clients. This guide is available on our website for your reference. If you have any feedback or suggestions regarding the methods we provide, please feel free to contact us directly. Our secretary is always here to assist you.
After preparing the cell suspension, Shanghai Jinma calculates the number of cells present, typically expressed as cell count per milliliter (cells/mL).
**Materials Needed:** - Cell suspension - Counting chamber (hemocytometer) - Microscope
**Procedure:** 1. Clean the counting chamber with alcohol and dry it thoroughly. 2. Place the cover slip over the counting chamber and slightly lift it to allow the cell suspension to be added. 3. Use a pipette to draw up the cell suspension from the culture flask and mix gently. 4. Carefully add the cell suspension drop by drop along the edge of the cover slip, ensuring that the space between the counting plate and the cover slip is filled without creating air bubbles or spilling over the edges.
**Counting Cells:** Under a microscope, count the cells in the four corners of the grid. Only count cells that are on the left and top lines; exclude those on the right and bottom lines to avoid double-counting.
**Calculation Formula:** Cell number / mL = (Total cell count) × (Dilution factor) × (10,000)
**Notes:** - Occasionally, multiple cells may cluster together. In such cases, treat them as a single unit. - If more than 10% of the cells appear in clusters, it may indicate incomplete digestion. - If the cell count is less than 200 cells/10 mm² or exceeds 500 cells/10 mm², the dilution may be incorrect. It’s recommended to reprepare the cell suspension and recount.
**Cryopreservation of Cultured Cells** Adding a cryoprotectant like dimethyl sulfoxide (DMSO) to the culture medium helps protect cells from ice crystal damage and osmotic stress during freezing. This allows long-term storage of cells in liquid nitrogen at -190°C.
**Materials Required:** - Cryopreservation tube - Cryoprotectant (e.g., DMSO or glycerol) - Culture medium - Fetal bovine serum (FBS) - Liquid nitrogen tank
**Steps for Cryopreservation:** 1. Harvest cells during the logarithmic growth phase. 2. Mix the cells with a cryopreservation solution containing 10–20% FBS. 3. Adjust the cell concentration to 1–2 million cells/mL. 4. Transfer the cell suspension into a cryotube and label it. 5. Freeze the cells slowly at a rate of 1–10°C/min. 6. If no programmable freezer is available, place the tubes in a 4°C refrigerator for 4 hours, then transfer to the gas phase of a liquid nitrogen container for 1 hour before submerging them in liquid nitrogen.
**Cell Recovery Process** To recover cryopreserved cells, follow these steps: 1. Remove the cryotube from the liquid nitrogen and immediately immerse it in a water bath at 37°C. 2. Gently shake the tube until the contents are fully thawed (within 20–60 seconds). 3. Open the tube under aseptic conditions and transfer the cells to a culture flask. 4. Add fresh culture medium and incubate at 37°C for 24 hours. 5. Replace the culture medium to remove the cryoprotectant and continue the culture.
**Transportation of Cells** For shipping, two main methods are used: 1. **Liquid Nitrogen/Dry Ice Method:** Requires specialized containers, which can be cumbersome and not ideal for long-term transport. 2. **Liquid-Filling Method:** A simpler and widely used technique that involves filling the culture vessel with enough medium to maintain cell viability during transit.
**Shanghai Jinma's Recommended Transport Method:** 1. Select cells in good condition, ideally after reaching near confluence. 2. Remove the old culture medium and replace it with fresh medium. 3. Fill the culture vessel to the neck, seal it securely, and leave a small amount of air inside. 4. Avoid excessive air bubbles, as they can negatively affect cell survival during transport. 5. Cells transported this way can remain viable for up to 4–5 days. Prolonged transport may reduce cell viability. Upon arrival, pour out most of the medium and retain only what is necessary to support cell growth. Incubate at 37°C and proceed with the next passage the following day.
We hope this detailed guide helps you better understand the processes involved in cell preservation, transportation, and recovery. If you need further assistance, don’t hesitate to reach out!
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