Strategies for Passaging and Culturing H22 Hepatocellular Carcinoma Cells from Ascitic Fluid

H22 cells (mouse hepatocellular carcinoma cells) are isolated from mouse ascites and established by Dalian Medical University. In experimental research, H22 cells are often implanted subcutaneously in mice to establish ectopic transplantation models, widely used for preliminary screening of anti-tumor drug efficacy.

Basic Information of H22 Cells:

• Growth Characteristics: Suspension cells
• Cell Morphology: Lymphoblast-like
• Growth Medium: RPMI-1640(PM150110) + 10% FBS + 1% P/S(PB180120)
• Recommended Seeding Density: 3×10^5-5×10^5 cells/mL
• Recommended Medium Change Frequency: 2-3 times per week
• Freezing Medium: 55% basal medium + 40% FBS + 5% DMSO or General Freezing medium(PB180436)
• Culture Conditions: Atmospheric: Air, 95%; CO2, 5%; Temperature: 37℃

Cell Passage and Culturing from Ascitic Fluid

Now that we've understood the basic information about H22 cells, we can proceed with cell culturing. Pricella has outlined detailed steps for ascitic fluid cell passage, hoping to be helpful for everyone!

1. After culturing H22 cells to the appropriate density, collect the cells by centrifugation (1200 rpm, 3 min) and resuspend them in PBS.

2. Take 0.2 mL of cultured cells (with a density of 1×10^7 cells/mL) and inject them into the mouse peritoneal cavity.

3. House the mice for 5-6 days until ascitic fluid can be collected (4-8 mL).

4. Aseptically collect the ascitic fluid by either aspiration or dissection, dilute it two fold with PBS(PB180327), centrifuge (1200 rpm, 3 min), discard the supernatant, repeat the washing process 1-2 times, and resuspend the cell pellet in 10 mL of PBS.

5. In a 50 mL centrifuge tube, layer 10 mL of mouse peripheral blood lymphocyte separation fluid, add 10 mL of cell suspension to the top layer, centrifuge (2000 rpm, 10 min), and repeat 2-3 times.

6. Resuspend the collected pellet in 1640 medium, seed into T175 culture flasks at a density of 4×10^5 cells/mL, and allow to incubate in a CO2 incubator for 8-12 hours.

7. Collect the cell suspension by centrifugation (1200 rpm, 3 min), and freeze the cells using a programmed freezing medium at a density of 10^7 cells/mL.

Points to Note

Mastering the aforementioned culturing steps doesn't guarantee successful cell culture. You also need to be aware of the following points:

1.Beginners can opt to euthanize the mice, withdraw some cells using a syringe, then open the abdomen and aspirate the remainder using a pipette. With proficiency, you can skip euthanasia and directly use a syringe to aspirate, allowing the mouse to be reused for subsequent injections.

2.Be careful not to puncture internal organs, as this can lead to cross-contamination of cells.

3.Ideally, house mice for 5 days. If housed for too long, mice may die, or the collected ascitic fluid may have high amount of red blood cells.

4.After revival, there may be dead cells, but this doesn't affect normal ascitic fluid production.

5.H22 cells need ascitic fluid passage every few generations during in vitro culture, otherwise, cell condition deteriorates rapidly, and fragmentation increases. For experiments, expand to the required cell quantity in advance and freeze for long-term culture, with ascitic fluid passage required every 5 generations.

6.After each passage, cells can be centrifuged, resuspended in PBS or saline, then injected into the next mouse.

7.If there's an excess of red blood cells in the collected ascitic fluid, red blood cell lysis solution can be used for removal.

8.During cell separation using separation fluid, multiple separations and screenings ensure cell purity.

9.For cell freezing, it's best to culture for around 8 hours after separation to effectively remove any residual red blood cells and optimize cell condition.

10.Cell vitality and the time it takes for ascitic fluid to develop vary based on factors like cell seeding density. Successful injection can be confirmed by observing mouse behavior; mice injected with ascitic fluid may show decreased activity.

Common Questions and Answers

Regarding H22 cell ascitic fluid passage, Pricella has summarized the most frequently asked questions as follows:

1.Which mice can be used for H22 ascitic fluid passage?

Balb/c mice, Km mice, etc., weighing around 20 g.

2.What precautions should be taken when receiving H22 cells?

Upon arrival, allow the cell vial to stand upright for 2-4 hours, then carefully remove the upper portion of the medium from the vial, leaving about 3mL of medium along with the cell pellet at the bottom of the vial. Centrifuge the aspirated cells at 1200 rpm for 3 minutes to collect the cell pellet.

3.Why do newly arrived H22 cells sometimes form cell aggregates?

During shipment, suspension cells should not be at too low a density to maintain stability. Due to the unstable condition during transportation, cell aggregates may form upon arrival. After stable culturing for a few days, the aggregates will disappear. It's important to handle the cells gently during culturing to avoid damaging them.