The development of antibody therapeutics is a highly complex process, and the establishment of stable, high-expression cell lines suitable for industrial production serves as the starting point and foundation for process development. A stable, high-yield engineered cell line not only significantly increases yield per unit volume and reduces production costs but also simplifies the downstream purification process, ensuring the production of safe, high-quality protein products.

I. Principles of Stable Cell Line Construction

    The advantage of stable cell lines is that they enable the sustained and stable expression of the target gene or the suppression of specific gene expression. This overcomes the limitation of short-lived target gene retention in transiently transfected cells, facilitating long-term observation of protein-protein interactions and the effects of genes on cellular function. The principle behind the construction of stable cell lines involves cloning the target gene into a vector containing a resistance gene. Commonly used resistance genes include neomycin, blasticidin, puromycin, or G418. The vector is then transfected into host cells, causing the target gene to integrate into the host cell chromosome, after which the mixed positive clones are screened using the resistance gene. In some cell lines, transfection efficiency is relatively low, and protein expression in mixed positive clones is reduced or inconsistent and not readily apparent. In such cases, it is necessary to isolate positive monoclonal cells from the mixed positive clones and subsequently establish a stably transfected cell line.

II. Methods for Constructing Stable Cell Lines

2.1 Lentiviral Infection

The lentiviral system is derived from the HIV-1 virus and can efficiently transfect both proliferating and non-proliferating cells, thereby introducing the target gene into various cell lines. The principle of the lentiviral infection system is that once the lentiviral vector enters the cell, it is reverse-transcribed into DNA in the cytoplasm. Upon entering the cell nucleus, it integrates randomly into the cellular genome, allowing for the expression of the target protein. This process enables the generation of stable cell lines expressing the target gene via a resistance gene.

2.2 Methods for Constructing Stable Cell Lines

(1) Cell screening (to verify cell health and check for mycoplasma contamination, etc.).

(2) Lentiviral packaging and purification: This requires a shuttle plasmid (carrying the target gene), one or two packaging plasmids (encoding viral core proteins, enzymes required for replication, and gene expression regulators, etc.), and an envelope plasmid (encoding the protein envelope). Prepare a transfection system, transfect the plasmids into 293T cells, and concentrate the viral supernatant via ultrafiltration or precipitate it by ultracentrifugation.

(3) Viral Transduction and Drug Screening: Determine the optimal MOI for the cells in advance, then infect seeded cells with the viral supernatant. If the lentivirus contains a fluorescent protein, distinct fluorescent expression is typically observable 48 hours after transfection. Begin drug screening of the cells 3–4 days after transfection.

Note: To achieve optimal experimental results, it is recommended to conduct preliminary experiments with 3–4 different MOI values before the formal experiment. It is also recommended to determine the optimal concentration in advance for different antibiotics used on the same cell type or for the same antibiotic used on different cell types.

(4) Detection of Stable Cell Lines: Detect at the mRNA level. After passing the detection, expand the cell line and cryopreserve it for long-term storage.

    The above steps outline the procedure for establishing a polyclonal stably transformed cell line. If a monoclonal stably transformed cell line is required, the following additional steps must be performed:

① Perform flow cytometry sorting on the screened polyclonal stably transformed cell line to isolate monoclonal cells;

② After the single cells have proliferated, proceed to a second round of screening;

③ Upon completion of the screening, collect a portion of the cells for mRNA level detection, and select the monoclonal cells with correct results for cryopreservation and strain preservation.

2.3 Advantages of Lentivirus-Mediated Stable Cell Line Construction

(1) Highly efficient transfection of host cells, resulting in high and stable levels of exogenous gene expression;

(2) Broad host range, capable of infecting almost all cell types (including both dividing and non-dividing cells), making it particularly suitable for cells with low plasmid transfection efficiency;

(3) Applicable to different cell types and various experimental purposes;

III. General Service Workflow for KMD Bioscience’s Stable Cell Line Construction Service

3.1 Vector Construction & Virus Packaging

    Generally, the humanized antibody gene is cloned into a lentiviral vector, followed by verification of plasmid expression. Lentiviral particles overexpressing the target gene are then obtained through packaging. The packaging components of the lentiviral vector system are transfected into 293 cells together with the vector components for packaging; after 1–2 days of cell culture, the cell supernatant is collected, concentrated, and tested for titer.

3.2 Cell Transfection

    First, determine the type of antibiotic, screening concentration, and viral transfection concentration. Transfect cells using the viral suspension, screen for stable transfected clones using antibiotics, and perform ELISA and Western blot (WB) analysis for identification.

3.3 Monoclonal Selection

    A cell pool is obtained immediately after transfection. Stably transfected positive clones can first be selected using a resistance marker, followed by screening the cell pool via the limited dilution method to isolate monoclonal lines, thereby obtaining a stably transfected monoclonal cell line.

3.4 Stable Cell Line Screening

    To rule out genetic instability, such as mutations, that may occur after multiple passages of the cell line, we select the 10 optimal clones. These 10 clones are passaged for 12–15 generations, and cell stability is assessed using methods such as qPCR and Western blot. Finally, we deliver the three cell lines with the best expression along with a detailed experimental report.

Other Services

For cells that cannot be passaged continuously, our company also offers cell immortalization services, followed by transfection and screening.

    This article is intended as a reference for science enthusiasts. It is not a substitute for professional expertise or practical experimental procedures that require more detailed and specialized information. If any content infringes on copyright, please contact the author to have the disputed material removed immediately.