Expert Insights | Tips For BV2 Cell Culture & Gene Editing

Expert Insights | Tips For BV2 Cell Culture & Gene Editing

BV2 cell line is an immortalized mouse-derived microglial cell line established by E. Blasi through retroviral-mediated transfection of v-raf/v-myc into mouse brain microglial cells[1]. This cell line retains many characteristics of microglial cells.

The BV2 cell line is of great value in research on immune responses, inflammation, and neurodegenerative diseases in the nervous system [2]. Its cultivation and gene editing are foundational steps in many experiments. Today, we’ll shares some tips on BV2 cell culture and gene editing, hoping to help you avoid some detours in your experiments and make the process smoother.

Figure 1: Normal BV2 cells (100×)

The cells are mainly round and spindle shaped. Astrocytes are mostly suspended; Adherent cells may be round or fusiform, and sometimes have short tentacles or protrusions.

Figure 2: Poor cell growth (100×)

Abnormal cell morphology, such as detached cells, cell shape change, and increased filopodia, etc

1.Cell Thawing

1) Preparation: pipet 7 mL of complete medium into a centrifuge tube.

2) Thawing: Remove the cells from dry ice, holding the lid with forceps, quickly thaw cells in a 37°C water bath by gently swirling the vial (Note: keep the cap out of the water). In about 1 minute, it would completely thaw. Stop the water bath until the ice melts to the size of green beans.

3) Centrifugation:Transfer the thawed cell suspension to a centrifuge tube and centrifuge at 1100 rpm for 4 minutes, discard the supernatant;

4) Resuspension and Seeding: Resuspend the cells with 1 mL of fresh complete medium and then transfer to appropriate culture flasks or culture dishes;

5) Cell Culture: Place the culture plates or flasks in a 37℃ incubator and observe the cell status after 24 hours.

2.Cell Passaging (for T25 flask):

1) When the cells are 70-80% confluent, it is ready to passage. Inside the ultra-cleanbench, remove and discard the medium from the flask and briefly rinse the cell 1-2 times with 5 mL PBS;

2) Add 1 mL of trypsin solution and allow it completely cover the cells, place the flask into the incubator and incubate for 1-2 mins, until the majority of the cells become round and lighter as observed under the microscope, a large number of cells detached from each side when gently shaking the flask, terminate trypsin digestion immediately;

3) Add complete medium to stop digestion, the volume is 2 times of trypsin (i.e. 2 mL for T25 flask). Then transfer the cells into a 15 mL centrifuge tube;

4) Centrifuge at 1100 rpm for 4 mins, then discard the supernatant. Then resuspend the cells with fresh complete medium;

5) Passage the cells at 1:3-1:5 passage ratio and observe the cell status after 24 hours.

3.Cell Cryopreservation

1) Collect the cells: Same as procedures of cell passaging. Digest the cells and transfer to a centrifuge tube;

2) Centrifuge: Centrifuge at 1100 rpm for 4 mins, then discard the supernatant;

3) Resuspension and Cryopreservation: Resuspend the cells with fresh complete medium. Adjust the cells to the required density (1x10^6 cells/mL) and then transfer to cryovials, labeled with the cell name, source, cell passage number, and date of cryopreservation ;

4) Cooling and Storage: Place the cryopreservation tube in the programmed cooling box, then put the container in -80℃ freezers. Stay overnight, transfer the cryovials to liquid nitrogen for long-term storage.

Precautions for cell culture:

1. Pay attention to the color of the culture medium and replace with the fresh culture medium in time: BV2 cells are very sensitive to the pH value of the culture medium, and the over acidic or over alkaline culture medium may lead to poor cell condition or even the detachment.

2. Handling Suspension Cells During Passaging: When a higher proportion of suspension cells is present, be sure to collect them during passaging and medium changes. If adherent cells are abundant and active, suspended cells can be discarded.

3. Frequent Medium Changes Due to Rapid Growth: BV2 cells grow quickly and consume medium at a fast rate. Change the medium promptly if the cell density becomes high or the medium shows signs of yellowing to avoid deteriorating cell condition. Passaging should be done when the density reaches 80% to prevent overgrowth.

4. After cryopreservation and thawing, the adherence of BV2 cells will become worse, and most of them are in the state of suspended cells. After 48 hours of culture, the suspended cells and adherent cells should be collected for subculture, and the cells will take on two forms: polygonal adherent cells and suspended cells.

5. BV2 cells are prone to differentiation, and the differentiated cells adhere to the plate surface more firmly and are very difficult to digest. Try to shorten the digestion time and prevent cell differentiation. In case of cell differentiation, some cells can be washed down by gentle pipetting and collected together with suspended cells for culture. The cells that cannot be washed down can be discarded directly, so as to reduce the degree of cell differentiation.

Adjusting Cell Condition:

1. Medium and Serum: Ensure the correct basal medium is used and that an appropriate amount of serum is added. Serum concentration could be adjusted according to the cell condition.

2. Cell Culture Environment: Check that the culture temperature, humidity, and gas conditions are optimal and stable.

3. Do not use the media expired or stored for long periods. Freshly prepared complete medium should ideally be used within two weeks.

4. When perform cell passaging, be mindful of digestion time (around 1 minute) and trypsin concentration. Avoid over digestion or under-digestion, as both can damage the cells.

5. When passaging or changing the medium, gently pipette the cells to avoid mechanical damage. This helps maintain cell health and prevents undue stress.

Tips for Transfection of BV2 Cells

1. Ensure the cell condition is good, and the cells are in the logarithmic growth phase, with a cell density generally around 70-80%.

2. Pay attention to the digestion time of the cells to avoid over digestion, which can cause damage to the cells.

3. During the experiment, ensure the cells are the single cells by pipetting to avoid cell clumping.

4. Cell viability during the experiment should be ≥80%.

Tips for Electroporation Method

1. The amount of electroporation cells should be well controlled during electroporation, and then inoculated into the appropriate culture plate;

2. When using the electroporation method to carry out the experiment, it is necessary to ensure that the cell adhesion rate after electroporation is ≥ 70%;

3. When transfection with electroporation method, the experimental time should be well controlled, and the whole electroporation process should not be too long.

Figure 3: Post-electroporation BV2 Cells (100X)

Notes for Lentivirus Method:

1. During the experiment, the cell confluence should be between 30-40% before infection, and not to be too high.

2. Conduct a preliminary experiment before the formal experiment to find the optimal MOI; add Polybrene before infection.

3. Ensure that Polybrene is well mixed before use to guarantee its uniformity.

Single-cell Cloning for BV2 Cells:

1. Ensure that the cell status is normal and that there are few senescent cells prior to single-cell cloning. It is recommended to maintain the confluence of cells at around 70%.

2. During cloning, the cell viability should be ≥90%.

3. Conduct a preliminary experiment to find the appropriate cloning dilution gradient to avoid too low a proportion of single clones.

4. After diluting the cell count, the results should preferably fall between 1*10^6-2*10^6.

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References

[1] Blasi, E et al. “Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus.” Journal of neuroimmunology vol. 27,2-3 (1990): 229-37. doi:10.1016/0165-5728(90)90073-v

[2] Henn, Anja et al. “The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation.” ALTEX vol. 26,2 (2009): 83-94. doi:10.14573/altex.2009.2.83