Morphology, Growth, and Troubleshooting in BV2 Cell Culture
The BV2 cell line was established by E. Blasi in 1990 through the immortalization of mouse microglia cells, achieved by retroviral-mediated transfection with v-raf/v-myc. BV2 cells are widely used in the study of neurological diseases and related mechanisms due to their retention of various morphological, characteristic, and functional features of microglial cells. Immunohistochemistry results show that BV2 cells are positive for MAC1 and MAC2 and negative for MAC3, GFAP (glial fibrillary acidic protein), and galactocerebroside.
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When culturing BV2 cells, one of the most common issues is abnormal cell morphology, often accompanied by changes in cell shape and the formation of elongated structures, which can affect experimental outcomes. In this article, we’ve compiled BV2 cell culture methods, common problems, and solutions to help you start your experiments quickly and efficiently.
Growth Characteristics of BV2 Cells
1) Partial adhesion and suspension: The ratio of suspension?to adherent cells is not fixed.
2) Polymorphism:?Suspension?cells are round, while adherent cells can be round (full and smooth with bright edges), spindle-shaped (extending dark projections at both ends), or polygonal.
Culturing Methods
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Culture Conditions:
Freezing Medium: 55% basal medium + 40% FBS + 5% DMSO.
Temperature: Liquid nitrogen.
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BV2 Cell Passage
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1. Use a pipette to collect the culture medium (containing suspension?cells) from the culture flask and transfer it to sterile centrifuge tube A.
2. Rinse the cells with 1 mL PBS, then transfer the PBS to centrifuge tube A.
3. Add 1 mL of trypsin?to the culture flask, gently swirl to ensure all cells are covered, and place the flask in a 37°C incubator for dissociation?(the time may vary depending on cell growth but usually lasts 2–4 minutes). After 1 minute, check the cells under a microscope. If they haven’t fully detached, continue dissociation?until the cells round up and detach. Stop dissociation when cells are visibly rounded and slightly detached from the surface; do not tap the flask.
4. Add 3 mL of serum-containing medium to stop dissociation, gently pipette up and down to detach the cells into the medium, and pipette 5–10 more times to evenly disperse the cells, ideally into a single-cell suspension.
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5. Transfer the cell suspension to centrifuge tube A, then centrifuge at 1200 rpm for 3 minutes.
6. Discard the supernatant, resuspend the pellet in 2–3 mL of fresh medium, and divide the cells into new culture flasks or dishes according to the required passage ratio. Ensure the cells are evenly dispersed.
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Notes
BV2 cells exhibit both adherent and suspension?growth, with the ratio of these two types not fixed. When passaging, it is important to collect both suspension?and adherent cells. If the percentage of suspension?cells is less than 10% and the adherent cells have reached over 80% confluence, you may choose not to collect the suspension?cells during passage.
Cryopreservation of BV2 Cells
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Recommended Freezing Medium:
55% basal medium + 40% FBS + 5% DMSO or 90% FBS + 10% DMSO.
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Notes:
After receiving cells, it is advisable to culture them for 2–3 passages before cryopreservation to ensure cell stability. The recommended cryopreservation density is 2–3 × 10? cells/mL. As cryopreservation success rates can vary, it is recommended to freeze?cells during each passage and thaw one vial after freezing to confirm cell viability.
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Common Issues in BV2 Cell Culture and Solutions
Issue 1: The cells appear predominantly round after arrival, but become more spindle-shaped during later passages. Why is this happening?
The morphology of BV2 cells changes with cell density. These cells proliferate rapidly, and due to transportation shock, many cells contract, making them appear round upon arrival. As you passage them (typically at a 1:2 to 1:4 ratio) and culture them for 24 hours, the cells will predominantly appear spindle-shaped. As the culture reaches higher densities, round cells will gradually increase.
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Issue 2: Cells exhibit extensive elongation and the formation of numerous projections during passaging. How can this be adjusted?
After passaging at low density, BV2 cells tend to form long projections. In this case, collect suspension?and weakly adherent cells in a 15 mL centrifuge tube. For adherent cells, digest them with trypsin for approximately 1 minute before stopping digestion. Collect the detached cells (those with extensive projections or elongated structures that are difficult to digest). Then, count and passage the cells. Over time, cell morphology will improve. For cells that did not detach during digestion, you can increase cell density and repeat this process, collecting the rounder cells for further culture.
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Issue 3: Are the spindle-shaped cells a sign of differentiation?
BV2 cells are prone to differentiation when stimulated by LPS, but they do not spontaneously differentiate under standard culture conditions. There is no definitive agreement on which morphology indicates differentiation. Some studies report that differentiation leads to an increase in spindle-shaped cells, while others suggest that differentiated cells tend to be round. It is recommended to test specific markers to determine whether differentiation has occurred.
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