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VEGF, also known as vascular permeability factor (VPF), is a dimeric glycoprotein that binds to cell-surface VEGF receptors (VEGFRs), thereby activating intracellular tyrosine kinases and initiating a cascade of signaling events involved in angiogenesis. In mammals, the VEGF family consists of five members: VEGFA, VEGFB, VEGFC, VEGFD, and placental growth factor (PGF). Members of the VEGF family bind in an overlapping manner to three receptor tyrosine kinases (VEGFR1, VEGFR2, and VEGFR3). All of these receptors belong to the transmembrane tyrosine kinase receptor family and comprise three main regions: an extracellular domain, a transmembrane domain, and an intracellular C-terminal domain. VEGFR1 and VEGFR2 are primarily expressed on vascular endothelial cells; however, VEGFR3 expression is particularly prominent on lymphatic endothelial cells. Compared to VEGFR1, VEGFR2 exhibits stronger pro-angiogenic activity and higher tyrosine kinase activity. VEGF primarily mediates in vivo angiogenic responses through the activation of VEGFR2. Studies have demonstrated that VEGFR2 is highly expressed in various malignancies, including ovarian cancer, thyroid cancer, melanoma, and medulloblastoma. Consequently, over the past decade or so, VEGFR2 has emerged as a critical target for the development of anti-angiogenic cancer therapies, leading to the market approval of several drugs.  

Upon binding to its receptor, VEGF forms a homodimeric complex that induces a conformational change in the protein; this triggers the autophosphorylation of tyrosine residues and propagates signals downstream. Key signaling pathways involved include the Ras/Raf/MAPK and PI3K/AKT pathways. Activation of these pathways leads to the activation of transcription factors, disruption of the cell cycle, promotion of cell migration, maintenance of cell proliferation, and inhibition of apoptosis—processes that collectively drive tumor initiation and progression. Conversely, under hypoxic conditions, growing tumor cells can activate the HIF-1α protein, thereby inducing the transcriptional expression and secretion of VEGF-A. This secreted VEGF-A then binds to VEGFR2 receptors located on the vascular endothelial cells surrounding the tumor, stimulating angiogenic responses and ultimately driving tumor growth. Therefore, VEGFR-2 signaling can be inhibited—thereby achieving therapeutic effects—by antibodies binding to VEGF-A or VEGFR-2 proteins, or by small-molecule inhibitors suppressing VEGFR-2 receptor kinase activity.

The Membrane-Anchored VEGF Cell serves as the target cell for the ADCC bioassay effector cell (V variant [High Affinity]-Fcγ-NFAT Jurkat), effectively mimicking *in vivo* ADCC effects; the underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the Cellular Model for the ADCC Effect

Figure 2. Recombinant Membrane Anchored VEGF Cell stably expressing VEGF.

Figure 3. Dose Response of Bevacizumab in ADCC Bioassay Effector Cell V variant (High Affinity)-Fcγ-NFAT Jurkat(C36) with Membrane Anchored VEGF Cell(C9).

Cell Resuscitation
1)Rapidly thaw the frozen cells in a 37 °C water bath for approximately 60 seconds. Once thawed (which may take slightly less or more than 60 seconds), immediately transfer the cell suspension from the cryovial into a 15 mL centrifuge tube containing 10 mL of pre-warmed  CHO-K1 Human Membrane Anchored VEGF Cell complete culture medium.
2)Centrifuge cells at 1000 rpm for 5 min to remove medium, then resuspend cells in 5 mL of pre-warmed complete medium.
3)Transfer the cell suspension into a T25 culture flask and incubate at 37 °C with 5% CO₂.
4)After approximately 24–36 hours, replace the medium or passage the cells to remove non-adherent dead cells.

Subculturing procedure
1)When the cell density reaches the appropriate confluency for passaging, wash the cells with PBS, then add 1 mL trypsin to detach the cells. When more than 80% of the cells detach upon gently tapping the culture flask, add complete culture medium to terminate digestion. Gently pipette to obtain a single-cell suspension, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.

2)Discard supernatant after centrifugation. Resuspend cells in fresh medium to a single-cell suspension and transfer to a new culture flask for continued growth.

Cell Freezing
After trypsinization and centrifugation of cells from each T75 flask or 10 cm culture dish, discard the supernatant. Add 2 mL of cryopreservation medium (90% FBS + 10% DMSO), gently resuspend thoroughly, and aliquot into two cryovials. Immediately place the cryovials into a controlled-rate freezing container (e.g., Nalgene 5100-0001), fill with isopropanol to the indicated level, and store at −80 °C. After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.