2012 月/24

KMD Bioscience-Nickel Affinity Chromatography

2024-12-20Protein EngineeringNickel Affinity Chromatographykmdbioscience

Nickel affinity chromatography is a type of immobilized metal ion affinity chromatography (IMAC) used for the purification of proteins that have been genetically engineered to include a polyhistidine tag (His-tag). His-tags are short sequences of histidine residues (usually 6–10) that bind strongly to nickel ions. This method is widely used in protein expression and purification systems because it is simple, efficient, and provides high purity of recombinant proteins.

Principle of Nickel Affinity Chromatography

Nickel affinity chromatography exploits the affinity between histidine residues in a His-tag and Ni²⁺ ions immobilized on a resin. The nickel is coordinated with nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) groups, which are covalently attached to the resin. The His-tagged protein binds to the nickel ions through the nitrogen atoms in the imidazole ring of the histidine residues, allowing selective retention of the target protein on the column, while other proteins flow through.

Steps of Nickel Affinity Chromatography

Preparation of the Column and Buffer Solutions

Resin Preparation: Nickel affinity chromatography uses resins such as Ni-NTA (nickel-nitrilotriacetic acid) or Ni-IDA (nickel-iminodiacetic acid) agarose.

Ni-NTA provides four coordination sites for nickel ions, making it more stable compared to Ni-IDA, which has three coordination sites.

Equilibration Buffer: This buffer is typically composed of:

50 mM Tris or phosphate buffer (pH 7.4–8.0): Maintains a stable pH.

150–500 mM NaCl: Helps maintain ionic strength and stability.

10–20 mM imidazole: Added to the buffer to reduce non-specific binding of host cell proteins, but still allows His-tagged proteins to bind to the resin.

Elution Buffer: The same buffer as the equilibration buffer, but with a higher concentration of imidazole (usually 200–500 mM) to elute His-tagged proteins by competing with the histidines for binding to nickel.

Loading the Protein Sample

The cell lysate or crude protein extract containing the His-tagged protein is first clarified by centrifugation or filtration to remove insoluble debris.

The clarified lysate is then applied to the equilibrated Ni-NTA or Ni-IDA column.

His-tagged proteins bind to the nickel ions on the resin, while other proteins lacking a His-tag are washed away.

Washing the Column

Wash the column with 5–10 column volumes of wash buffer containing a low concentration of imidazole (10–30 mM) to remove weakly bound proteins and non-specific contaminants.

The imidazole concentration must be optimized depending on the protein of interest. Too little imidazole may lead to non-specific binding, while too much may cause premature elution of the target protein.

Elution of His-Tagged Protein

After washing, the His-tagged protein is eluted by increasing the concentration of imidazole in the elution buffer (200–500 mM). Imidazole competes with the histidine residues in the His-tag for binding to the nickel ions, releasing the His-tagged protein from the resin.

The eluted protein fractions are collected, and the concentration of imidazole in the elution buffer can be varied to fine-tune the elution of the target protein.

Buffer Exchange or Desalting

If required, the eluted protein can be further processed by dialysis or desalting columns to remove imidazole, which can interfere with downstream applications such as enzyme assays or protein crystallization.

The final protein is then stored in an appropriate buffer, such as phosphate-buffered saline (PBS) or Tris-HCl, depending on the application.

Detailed Protocol for Nickel Affinity Chromatography

Materials

Ni-NTA or Ni-IDA resin

Equilibration buffer: 50 mM Tris-HCl or 50 mM phosphate buffer, 150 mM NaCl, 10–20 mM imidazole, pH 7.4–8.0

Wash buffer: Same as equilibration buffer, with 20–50 mM imidazole

Elution buffer: Same as equilibration buffer, with 200–500 mM imidazole

Cell lysate containing His-tagged protein

Column: Either gravity-flow columns or FPLC-compatible columns

pH meter, Centrifuge, Filter units (0.45 µm)

Imidazole, NaCl, Tris or phosphate buffer

1. Preparation of the Resin

Equilibrate the resin: If using a pre-packed column or gravity-flow column, equilibrate the resin with 5–10 column volumes of equilibration buffer.

If using loose resin for batch purification, slurry the resin with equilibration buffer and transfer to the column, then equilibrate as above.

2. Sample Preparation

Prepare the cell lysate: Lyse the cells (bacterial, yeast, or mammalian cells) by sonication, freeze-thaw cycles, or detergent-based lysis (e.g., using Triton X-100 or NP-40).

Clarify the lysate: Centrifuge the lysate at high speed (e.g., 15,000 x g for 15–20 minutes) to remove debris. Filter the supernatant through a 0.45 µm filter to avoid clogging the column.

Check the pH: Ensure that the pH of the lysate is close to neutral (pH 7.4–8.0) for optimal binding.

3. Loading the Sample

Apply the clarified lysate to the equilibrated Ni-NTA or Ni-IDA column.

Flow rate: For gravity-flow columns, use a slow flow rate (~1 drop/second) to allow maximum interaction between His-tagged proteins and the resin. For FPLC, use a recommended flow rate depending on column capacity.

Collect the flow-through (non-binding fraction) and retain for analysis to check if any target protein is lost.

4. Washing the Column

Wash the column with 5–10 column volumes of wash buffer containing 20–50 mM imidazole. This removes non-specifically bound proteins while retaining His-tagged proteins on the column.

Collect the wash fractions for analysis (e.g., by SDS-PAGE) to monitor the removal of contaminants.

5. Elution

Elute the bound His-tagged protein using elution buffer containing a higher concentration of imidazole (200–500 mM).

Collect fractions during elution and monitor the protein elution using UV absorbance (e.g., at 280 nm) or by performing SDS-PAGE to identify the fractions containing the target protein.

If the protein does not elute efficiently, increase the imidazole concentration or extend the elution time.

6. Analysis and Storage

Analyze the purity of the eluted fractions by SDS-PAGE or Western blotting.

If needed, pool the fractions containing the target protein and concentrate or dialyze the protein to remove imidazole or exchange the buffer for downstream applications.

Troubleshooting Tips for Nickel Affinity Chromatography

Low Yield

Cause: Improper binding of the His-tagged protein to the column.

Solution: Check that the pH of the buffer is in the optimal range (pH 7.4–8.0). Ensure that the His-tag is not blocked (e.g., ensure it’s not cleaved off or inaccessible).

Poor Purity

Cause: Non-specific proteins binding to the column.

Solution: Increase the imidazole concentration in the wash buffer (e.g., from 10 mM to 30 mM) to reduce non-specific binding without affecting His-tagged protein binding.

Protein Doesn’t Elute

Cause: Strong binding of the His-tagged protein to the nickel resin.

Solution: Increase the imidazole concentration in the elution buffer (up to 500 mM). Alternatively, you can use EDTA (ethylenediaminetetraacetic acid) to strip the nickel from the resin, but this may co-elute unwanted proteins or damage the resin for reuse.

Protein Precipitation

Cause: Imidazole concentration or pH change during elution.

Solution: Include a stabilizer in the buffer (e.g., glycerol or reducing agents like DTT) to prevent precipitation. Avoid rapid pH changes or high imidazole concentrations that might destabilize the protein.

Advantages of Nickel Affinity Chromatography

High Specificity: The interaction between His-tags and Ni²⁺ ions is highly specific, leading to selective binding of His-tagged proteins.
Simplicity: The procedure is straightforward and relatively quick, allowing for rapid purification.
Scalability: Nickel affinity chromatography can be scaled from small laboratory volumes to industrial-scale protein purification.
Reusability: Ni-NTA resin can be regenerated and reused multiple times, making it cost-effective.

Disadvantages

Non-Specific Binding: Low concentrations of imidazole in the buffer may lead to non-specific binding of other proteins.
Imidazole Contamination: High concentrations of imidazole in the elution buffer can interfere with downstream applications, requiring additional steps for removal.
Tag Dependence: The method requires the presence of a His-tag, which may not always be practical for native protein purification.

Applications

Recombinant Protein Purification: Used extensively for purifying His-tagged proteins expressed in bacterial, yeast, or mammalian systems.

Enzyme Assays: Purified proteins can be used for biochemical assays and enzymatic activity studies.

Structural Biology: High-purity His-tagged proteins are often required for X-ray crystallography or NMR studies.

Immunoprecipitation: Used to isolate His-tagged proteins from complex mixtures for further analysis.

Nickel affinity chromatography is a widely-used technique for the purification of recombinant His-tagged proteins due to its high efficiency, specificity, and versatility.

1812 月/24

KMD Bioscience-Antibody Affinity Maturation

2024-12-18Antibody DiscoveryAntibody Affinity Maturationkmdbioscience

Antibody affinity maturation is the process by which B cells produce antibodies with increasing affinity for their specific antigen over time, especially during an immune response. This process occurs primarily in the germinal centers of lymphoid tissues, such as the spleen and lymph nodes, and involves somatic hypermutation (SHM) and clonal selection of B cells.

Key Steps in Antibody Affinity Maturation

Activation of B Cells

Naive B cells are activated when their B cell receptor (BCR), membrane-bound immunoglobulin, binds to an antigen. This interaction typically occurs in lymphoid organs, where antigens are presented by follicular dendritic cells (FDCs) or other antigen-presenting cells (APCs).

After antigen binding and additional co-stimulation from helper T cells, naive B cells proliferate and migrate into the germinal centers of lymphoid tissues.

Somatic Hypermutation (SHM)

Once in the germinal center, B cells undergo somatic hypermutation, a process mediated by the enzyme activation-induced cytidine deaminase (AID).

SHM introduces point mutations at a high rate in the variable regions of the immunoglobulin (Ig) genes, particularly in the regions coding for the antigen-binding sites.

These mutations are random, and they either increase, decrease, or have no effect on the antibody’s affinity for the antigen.

Clonal Selection

Following somatic hypermutation, B cells with different affinities for the antigen are generated. The B cells that produce antibodies with higher affinity for the antigen are preferentially selected.

B cells compete for limited amounts of antigen presented by follicular dendritic cells (FDCs) and for help from T follicular helper (Tfh) cells.

B cells with higher affinity BCRs are better able to capture antigen from FDCs and present antigen to Tfh cells, resulting in stronger survival and proliferation signals.

B cells with low-affinity BCRs, or those with detrimental mutations, are outcompeted and undergo apoptosis.

Class Switch Recombination (CSR) (Optional but Related)

In parallel with affinity maturation, B cells may undergo class switch recombination, where the constant region of the antibody heavy chain changes (e.g., from IgM to IgG, IgA, or IgE), allowing the antibody to acquire different effector functions without changing its antigen specificity.

Differentiation into Plasma Cells and Memory B Cells

B cells with high-affinity BCRs exit the germinal center and differentiate into plasma cells or memory B cells.

Plasma cells secrete large amounts of high-affinity antibodies to combat the current infection, while memory B cells provide long-lasting immunity, allowing for a quicker and stronger immune response if the antigen is encountered again in the future.

Molecular Mechanisms of Affinity Maturation

Somatic Hypermutation (SHM)

AID (Activation-Induced Cytidine Deaminase) is the key enzyme involved in SHM. It deaminates cytidine residues in the variable regions of the immunoglobulin genes, converting them into uracil.

DNA repair mechanisms then process these uracil residues, introducing mutations. These mutations are often focused on the complementarity-determining regions (CDRs) of the immunoglobulin genes, which directly interact with the antigen.

SHM creates a pool of B cells with antibodies of varying affinities for the antigen.

Clonal Selection

Only B cells with the highest-affinity BCRs receive sufficient survival signals through interactions with antigen-presenting cells and Tfh cells.

B cells with high-affinity BCRs present more antigen peptides to Tfh cells, which provide survival and proliferation signals through cytokines like IL-4 and IL-21.

Importance of Antibody Affinity Maturation

Increased Binding Strength

Affinity maturation ensures that antibodies produced later in an immune response bind more tightly and specifically to their target antigen. This is critical for neutralizing pathogens effectively.

Improved Immune Defense

Higher-affinity antibodies are better at neutralizing pathogens, opsonizing microbes for phagocytosis, and activating the complement system, leading to more efficient clearance of the pathogen.

Vaccine Efficacy

Vaccines are designed to stimulate affinity maturation, so that the immune system generates memory B cells capable of producing high-affinity antibodies upon re-exposure to the pathogen.

Autoimmunity Risk

While affinity maturation is crucial for an effective immune response, it also carries the risk of generating autoreactive B cells due to the random nature of SHM. Mechanisms such as negative selection and regulatory checkpoints are in place to eliminate autoreactive B cells, but breakdowns in these processes can lead to autoimmune diseases.

Experimental Approaches for Studying Affinity Maturation

ELISA (Enzyme-Linked Immunosorbent Assay)

ELISA is used to measure the affinity of antibodies produced by B cells in response to antigen stimulation. It quantifies the binding strength of antibodies to a specific antigen over the course of an immune response.

Surface Plasmon Resonance (SPR)

SPR is a technique that allows for the real-time measurement of antibody-antigen binding affinities, providing detailed insights into the kinetics of affinity maturation.

Next-Generation Sequencing (NGS)

NGS can be used to sequence the immunoglobulin genes of B cells from germinal centers or other immune tissues. This provides information on the accumulation of mutations in the variable regions of the antibody genes during affinity maturation.

Single-Cell RNA Sequencing

Single-cell RNA sequencing can be used to study the transcriptional profiles of individual B cells undergoing affinity maturation in the germinal center, allowing researchers to identify key molecular pathways involved in the process.

B Cell Receptor (BCR) Sequencing

BCR sequencing is used to track the evolution of B cell clones during affinity maturation by analyzing the somatic mutations in the variable regions of the immunoglobulin genes.

Clinical and Therapeutic Implications

Monoclonal Antibody Development

Affinity maturation can be mimicked in vitro to create high-affinity monoclonal antibodies for therapeutic purposes, such as cancer immunotherapy, autoimmune disease treatment, or infectious disease management.

Vaccine Design

Understanding the mechanisms of affinity maturation helps in the design of vaccines that promote long-lasting immunity by stimulating the production of high-affinity antibodies.

Autoimmune Disease

Aberrations in affinity maturation can lead to the development of autoreactive antibodies, contributing to autoimmune diseases like lupus and rheumatoid arthritis. Therapies targeting autoreactive B cells aim to correct these issues.

In summary, antibody affinity maturation is a critical process that enhances the immune system’s ability to produce highly specific and effective antibodies during an immune response. It involves somatic hypermutation in the germinal centers, followed by clonal selection, where B cells producing higher-affinity antibodies are selected to survive and proliferate. This process is vital for effective immunity, vaccine responses, and therapeutic antibody development.

1312 月/24

KMD Bioscience-EBV Immortalized B Cells

2024-12-13CRO ServiceEBV Immortalized B Cellskmdbioscience

Epstein-Barr virus (EBV)-immortalized B cells, also known as lymphoblastoid cell lines (LCLs), are widely used in research for various applications, particularly in studies of human genetics, immunology, and infectious diseases. EBV immortalization allows B cells to proliferate indefinitely while maintaining many of their functional properties, such as antibody production and the ability to present antigens.

Mechanism of EBV-Mediated Immortalization

Infection with EBV

Epstein-Barr virus, a member of the herpesvirus family, infects B lymphocytes by binding to the CD21 receptor on their surface.

Once inside the cell, EBV establishes a latent infection. During latency, the virus expresses several proteins, including Epstein-Barr Nuclear Antigen 2 (EBNA-2) and Latent Membrane Protein 1 (LMP-1).

Cell Cycle Regulation

EBNA-2: Activates the transcription of viral and cellular genes that are essential for B cell proliferation.

LMP-1: Mimics the signaling of CD40, a crucial molecule in B cell activation. This signaling prevents apoptosis and promotes continuous cell division.

Immortalization

The expression of EBV genes drives the B cell into a state of continuous proliferation, allowing it to divide indefinitely while retaining many functional characteristics of primary B cells.

EBV Immortalization Protocol

Materials

Peripheral blood mononuclear cells (PBMCs) or purified B cells

Epstein-Barr Virus (EBV)-containing supernatant or EBV-producing cell line (e.g., B95-8)

RPMI-1640 medium (or other appropriate growth medium for B cells)

Fetal bovine serum (FBS)

Penicillin-Streptomycin

Phytohemagglutinin (PHA, optional)

Cyclosporine A (optional, to suppress T cell activation)

37°C incubator with 5% CO₂

Step-by-Step Protocol

Isolation of PBMCs or B Cells

Collect blood from the donor and isolate peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation (e.g., using Ficoll-Paque).

Alternatively, purify B cells from the PBMCs using magnetic beads (CD19+ or CD20+ beads) if you require higher purity.

Infection with EBV

Seed the PBMCs or purified B cells in a 24-well or 6-well plate at a concentration of 0.5-1 million cells per well in RPMI-1640 medium supplemented with 10-15% FBS and penicillin-streptomycin.

Add the EBV-containing supernatant (typically from an EBV-producing cell line such as B95-8) to the wells. Use a 1:1 or 1:2 ratio of EBV supernatant to growth medium.

Optional: Add phytohemagglutinin (PHA) to a final concentration of 1-2 µg/mL to stimulate initial B cell activation.

Optional: Add cyclosporine A at a concentration of 0.5-1 µg/mL to inhibit T cell proliferation, which can interfere with B cell immortalization.

Incubation

Incubate the cells at 37°C in a 5% CO₂ incubator. After 24-48 hours, gently replace the medium with fresh RPMI-1640 containing 10-15% FBS, without removing the cells.

Continue changing the medium every 3-4 days, ensuring not to discard any of the adherent or semi-adherent B cells during the medium changes.

Growth and Expansion

After 1-2 weeks, clusters of proliferating B cells (lymphoblastoid cell lines) should start to form. These clusters will grow as LCLs, exhibiting rapid proliferation.

After 2-3 weeks, the cell culture should be expanded into larger flasks or plates as the LCLs continue to grow.

Monitor the culture for contamination or overgrowth of non-B cell populations, although the addition of cyclosporine A can help suppress contaminating T cells.

Cryopreservation

Once the immortalized B cells reach sufficient numbers, they can be cryopreserved in freezing medium (e.g., 10% DMSO in FBS) for long-term storage and future use.

Characterization of EBV-Immortalized B Cells

Phenotypic Confirmation

Confirm the immortalization by testing for the expression of B cell markers (e.g., CD19, CD20) via flow cytometry or immunofluorescence.

The cells should also express EBV latent proteins such as EBNA-2 and LMP-1, which can be detected by Western blotting or PCR.

Proliferation Assays

EBV-immortalized B cells should show continuous proliferation in culture. Proliferation assays, such as cell counting or MTT assays, can be used to measure their growth rate.

Functional Studies

EBV-immortalized B cells retain many functional properties of primary B cells, such as antibody production and antigen presentation. These cells can be used in immune response studies, including cytokine secretion, antigen processing, and antibody secretion assays.

Applications of EBV-Immortalized B Cells

Human Genetic Studies

EBV-immortalized B cells are commonly used in large-scale genetic studies because they provide a renewable source of DNA. Immortalized B cells are particularly valuable for genome-wide association studies (GWAS) and for studying inherited disorders.

Immunology and Infectious Disease Research

LCLs are useful for investigating the biology of B cells, including studies on antibody production, cytokine secretion, and antigen presentation.

Researchers use EBV-immortalized B cells to study how the virus interacts with the immune system and contributes to diseases such as autoimmune disorders and B cell lymphomas.

Vaccine Development

EBV-immortalized B cells are often used to study the development of vaccines targeting viral infections, as they provide insight into how the immune system responds to viral antigens.

Monoclonal Antibody Production

These immortalized cells can be used to generate B cell clones that produce specific monoclonal antibodies for therapeutic or diagnostic purposes.

Functional Assays

LCLs can be used in functional assays to study B cell signaling, antigen presentation, and their interaction with T cells or other immune cells. They are valuable in studies related to transplantation immunology or autoimmunity.

Cancer Research

Because EBV immortalization mimics certain steps in B cell transformation leading to lymphoma, these cells serve as a model for studying B cell malignancies, such as Burkitt’s lymphoma and Hodgkin’s lymphoma.

Advantages and Limitations of EBV-Immortalized B Cells

Advantages

Unlimited Proliferation: LCLs provide a continuous, renewable source of B cells.

Maintenance of B Cell Functions: These cells retain their ability to produce antibodies and present antigens, making them useful for functional immunology studies.

Long-Term Genetic Stability: EBV-immortalized B cells are a stable source of genetic material for research, providing consistent results over time.

Limitations

Viral Gene Expression: The presence of EBV genes (e.g., EBNA-2, LMP-1) may interfere with some cellular processes, potentially limiting the use of these cells in certain types of experiments.

Risk of Transformation: Although rare, EBV-immortalized B cells may undergo additional mutations over time, leading to transformation or cancer-like growth.

Variable Yield: The efficiency of EBV-mediated immortalization can vary, and some B cell populations may not immortalize as efficiently as others.

EBV-immortalized B cells are a powerful tool in immunology, genetics, and infectious disease research, providing researchers with a stable and renewable source of functional human B cells for long-term studies.

1212 月/24

KMD Bioscience-hTERT Immortalized Primary Cells

2024-12-12CRO ServicehTERT Immortalized Primary Cellskmdbioscience

hTERT-immortalized primary cells are cells that have been genetically modified to express the human telomerase reverse transcriptase (hTERT) gene, enabling them to bypass senescence and divide indefinitely. Unlike other immortalization techniques, hTERT overexpression maintains many of the original characteristics of primary cells, allowing for long-term culture without significant genetic changes.

Mechanism of hTERT Immortalization

Telomerase and Cellular Senescence

In most somatic cells, the enzyme telomerase is not active, leading to the progressive shortening of telomeres with each cell division. When telomeres become critically short, the cell enters senescence (growth arrest) or undergoes apoptosis.

hTERT is the catalytic subunit of the enzyme telomerase, which elongates telomeres, preventing their shortening and allowing cells to continue dividing indefinitely.

hTERT Overexpression

By introducing the hTERT gene into primary cells, researchers can reactivate telomerase, preventing telomere shortening and enabling cells to proliferate beyond their normal lifespan while preserving their normal physiological functions.

hTERT Immortalization Protocol

Here’s a generalized protocol for immortalizing primary cells with hTERT.

Materials

Primary cells (e.g., fibroblasts, epithelial cells)

Lentiviral or retroviral vector containing the hTERT gene

Lentiviral or retroviral packaging plasmids (if producing your own virus)

Transfection reagent (e.g., Lipofectamine 2000)

HEK 293T cells (for viral packaging, if producing your own virus)

Growth medium (e.g., DMEM or RPMI-1640)

Antibiotic for selection (e.g., puromycin or neomycin)

Polybrene (for enhanced transduction efficiency)

Incubator set to 37°C with 5% CO₂

Day 1: Virus Production (if not using pre-made virus)

HEK 293T Transfection

Seed HEK 293T cells (or another packaging cell line) at approximately 70-80% confluency in a 10 cm dish.

Transfect the cells with the hTERT-expressing vector and the packaging plasmids using a transfection reagent like Lipofectamine 2000 or PEI.

After 6-8 hours, replace the medium with fresh complete DMEM containing 10% FBS.

Harvest Lentiviral/Retroviral Supernatant

Collect the virus-containing supernatant from the transfected cells 48-72 hours after transfection.

Filter the supernatant through a 0.45 µm filter to remove cell debris.

Concentrate the viral particles using ultracentrifugation (optional) or use them directly for transduction.

Day 3: Transduction of Primary Cells

Prepare Primary Cells

Seed your primary cells (e.g., fibroblasts, epithelial cells) in a 6-well or 12-well plate at 50-70% confluency.

Lentiviral/Retroviral Transduction

Add 5-10 µg/mL of polybrene to the culture medium to enhance transduction efficiency.

Add the viral supernatant containing hTERT to the cells at an appropriate multiplicity of infection (MOI). A higher MOI will increase the likelihood of successful transduction.

Incubate the cells with the viral supernatant for 24 hours at 37°C.

Medium Replacement

After 24 hours, replace the viral supernatant with fresh complete medium and continue to culture the cells for 48-72 hours to allow for stable expression of hTERT.

Day 6: Selection of Transduced Cells (Optional)

Antibiotic Selection

If the hTERT-expressing vector contains an antibiotic resistance gene (e.g., puromycin or neomycin), begin selection by adding the appropriate antibiotic to the culture medium.

Use puromycin (1-2 µg/mL) or neomycin/G418 (400-800 µg/mL), depending on the vector.

Maintain selection for 3-5 days until non-transduced cells are eliminated and only antibiotic-resistant cells remain.

Expand Selected Cells

Once selection is complete, allow the surviving hTERT-expressing cells to expand in fresh medium. Change the medium every 2-3 days.

Day 10+: Validation and Expansion

Validate hTERT Expression

Confirm the expression of hTERT in the immortalized cells by using quantitative PCR (qPCR) or Western blotting.

Assess telomerase activity using the TRAP assay (Telomeric Repeat Amplification Protocol), which measures the activity of telomerase.

Proliferation and Senescence Assays

Perform proliferation assays (e.g., cell counting, MTT) to confirm that the hTERT-immortalized cells have an extended lifespan compared to non-immortalized cells.

Stain the cells for β-galactosidase (a marker of senescence) to ensure that the immortalized cells are not senescent.

Cryopreservation

Once the immortalized cells are proliferating stably, freeze them in freezing medium (e.g., 10% DMSO in FBS) for long-term storage.

Advantages of hTERT Immortalization

Preservation of Cell Properties: Unlike immortalization methods involving viral oncoproteins (e.g., SV40 or HPV), hTERT immortalization usually preserves the cells’ normal physiology, differentiation potential, and genomic stability.

Extended Proliferative Capacity: hTERT allows cells to proliferate indefinitely, making it possible to conduct long-term studies that would not be feasible with primary cells alone.

Low Risk of Tumorigenicity: Since hTERT immortalization does not directly interfere with tumor suppressor pathways (like p53 or pRb), the cells are less likely to become tumorigenic compared to other methods.

Examples of hTERT-Immortalized Cell Lines

BJ-hTERT (Human Foreskin Fibroblasts)

Derived from human foreskin fibroblasts, these cells are commonly used for studying fibroblast biology, skin aging, and wound healing.

The cells maintain typical fibroblast morphology and characteristics while being able to proliferate indefinitely.

N/TERT-1 and N/TERT-2 (hTERT-Immortalized Keratinocytes)

Human epidermal keratinocytes immortalized with hTERT, widely used to study skin biology, wound healing, and skin disorders.

These cells retain many of the characteristics of normal keratinocytes, including their ability to differentiate in response to calcium and other signals.

HUVEC-hTERT (Human Umbilical Vein Endothelial Cells)

These endothelial cells, immortalized with hTERT, are often used for studies of angiogenesis, vascular biology, and cardiovascular diseases.

The cells retain their ability to form tube-like structures in vitro, a characteristic of endothelial cells.

RPE1-hTERT (Retinal Pigment Epithelial Cells)

Retinal pigment epithelial cells immortalized with hTERT are used for research in ophthalmology, particularly for studying retinal degeneration and age-related macular degeneration (AMD).

MSC-hTERT (Mesenchymal Stem Cells)

Mesenchymal stem cells from bone marrow or adipose tissue are immortalized with hTERT to study stem cell biology, differentiation, and regenerative medicine applications.

Applications of hTERT-Immortalized Cells

Cancer Research

hTERT-immortalized cells are often used to study the role of telomerase in cancer development and to investigate how cancer cells avoid senescence.

Aging Research

These cells are essential for studying cellular aging, telomere biology, and the molecular mechanisms that drive cellular senescence.

Regenerative Medicine and Tissue Engineering

hTERT-immortalized cells serve as models for developing tissue-engineered constructs and studying stem cell differentiation and tissue repair processes.

Drug Discovery and Toxicology

Immortalized primary cells are valuable tools for high-throughput drug screening and evaluating the cytotoxicity or efficacy of new therapeutic compounds.

Basic Cell Biology

These cell lines provide a renewable, physiologically relevant model system for studying fundamental cellular processes, such as cell cycle regulation, DNA repair, and apoptosis.

hTERT immortalization is a powerful method to generate cell lines with extended proliferative capacity while maintaining many of the original characteristics of the primary cells. These cell lines are essential for a wide variety of research applications, including aging studies, cancer biology, and regenerative medicine.

1112 月/24

KMD Bioscience-SV40 Large T Antigen Immortalization

2024-12-11CRO ServiceSV40 Large T Antigen Immortalizationkmdbioscience

SV40 Large T antigen is a viral protein derived from the Simian Virus 40 (SV40), widely used for cell immortalization. It works by inactivating key tumor suppressor proteins like p53 and retinoblastoma protein (Rb), enabling cells to bypass senescence (the state in which cells stop dividing) and proliferate indefinitely. This method of immortalization has been extensively used in a variety of cell types, including fibroblasts, epithelial cells, and endothelial cells.

Mechanism of SV40 Large T Antigen Immortalization

Inhibition of p53

The p53 protein plays a crucial role in controlling the cell cycle, inducing apoptosis, and halting the cell cycle in response to DNA damage. SV40 Large T antigen binds to p53, inhibiting its function. This prevents cells from undergoing apoptosis and allows them to continue dividing even when they have accumulated mutations or DNA damage.

Inhibition of Retinoblastoma Protein (Rb)

Rb regulates the cell cycle by preventing cells from transitioning from the G1 phase to the S phase (DNA replication). SV40 Large T antigen binds to Rb and inactivates it, allowing cells to progress through the cell cycle uncontrollably and bypass growth suppression.

Cell Cycle Deregulation

By inhibiting both p53 and Rb, SV40 Large T antigen drives the cell into a state of continuous proliferation. This allows cells to evade the normal checkpoints that limit their lifespan and can result in immortalization.

Advantages of SV40 Large T Antigen Immortalization

Broad Applicability: SV40 Large T antigen can immortalize a wide range of cell types, making it a versatile tool for different kinds of research.

High Efficiency: This method is highly effective for inducing immortalization and bypassing senescence.

Established Protocols: SV40 Large T antigen has been used for decades, and well-established protocols exist for various cell types.

Limitations of SV40 Large T Antigen Immortalization

Genetic Instability: Since SV40 Large T antigen interferes with tumor suppressor pathways, immortalized cells may accumulate additional mutations over time, increasing the risk of transformation (tumor-like growth).

Altered Cell Function: Some cells may lose their normal phenotype or functional characteristics after immortalization due to changes in their cell cycle regulation.

Tumorigenic Potential: Cells immortalized by SV40 Large T antigen may become tumorigenic if implanted in vivo, making them unsuitable for certain studies.

SV40 Large T Antigen Immortalization Protocol

Materials

Primary cells (e.g., fibroblasts, epithelial cells)

Lentiviral or retroviral vector encoding SV40 Large T antigen

Lentiviral or retroviral packaging plasmids (for virus production, if required)

Transfection reagent (e.g., Lipofectamine 2000 or PEI)

HEK 293T cells (for producing lentivirus, if required)

Polybrene (to enhance transduction efficiency)

Antibiotic for selection (e.g., puromycin or neomycin)

Growth medium appropriate for your cell type (e.g., DMEM, RPMI-1640)

Incubator set to 37°C with 5% CO₂

Step-by-Step Protocol

Virus Production (if not using pre-made virus)

Transfection of Packaging Cells: Transfect HEK 293T cells (or another packaging cell line) with the SV40 Large T antigen-containing vector along with the packaging plasmids (for lentiviral or retroviral production).

Harvest Viral Supernatant: After 48-72 hours, collect the virus-containing supernatant from the transfected HEK 293T cells. Filter the supernatant using a 0.45 µm filter to remove cell debris.

Optional: Concentrate the viral supernatant using ultracentrifugation or a commercial viral concentration kit.

Transduction of Primary Cells

Prepare Primary Cells: Seed your primary cells (e.g., fibroblasts, epithelial cells) in a 6-well or 12-well plate at 50-70% confluency.

Lentiviral/Retroviral Transduction: Add polybrene (5-10 µg/mL) to the culture medium to enhance viral transduction. Then, add the viral supernatant containing SV40 Large T antigen to the cells. Incubate the cells with the virus for 24 hours.

Medium Change: After 24 hours, replace the viral supernatant with fresh medium.

Selection of Transduced Cells

Antibiotic Selection: If the vector contains an antibiotic resistance marker (e.g., puromycin or neomycin), add the appropriate antibiotic to the culture medium to select for successfully transduced cells. Use puromycin (1-2 µg/mL) or neomycin (400-800 µg/mL) as appropriate for your system.

Expand Surviving Cells: Allow the surviving cells to expand in fresh medium once non-transduced cells have been eliminated.

Validation of Immortalization

Confirm SV40 Large T Antigen Expression: Validate the expression of SV40 Large T antigen using Western blotting, immunofluorescence, or PCR.

Proliferation Assays: Confirm that the cells have an extended lifespan and can proliferate indefinitely by performing growth curve analysis.

Test for Senescence: Stain the cells for β-galactosidase, a marker of cellular senescence, to ensure that the immortalized cells are not senescent.

Cryopreservation

Once the immortalized cells have been validated, cryopreserve them in freezing medium (e.g., 10% DMSO in FBS) for future use.

Example of SV40 Large T Antigen Immortalized Cell Lines

COS-1 and COS-7

Cell Type: African green monkey kidney cells immortalized with SV40 Large T antigen.

Applications: These cell lines are widely used for transfection experiments, protein expression, and studying virus-host interactions.

HEK 293T

Cell Type: Human embryonic kidney cells, originally immortalized using SV40 Large T antigen.

Applications: HEK 293T cells are extensively used for viral packaging, transfection studies, and recombinant protein production.

MRC-5 SV40

Cell Type: Human lung fibroblasts immortalized with SV40 Large T antigen.

Applications: These cells are used in studies of lung biology, cancer research, and drug screening.

SVEC4-10

Cell Type: Mouse endothelial cells immortalized with SV40 Large T antigen.

Applications: These cells are commonly used to study endothelial function, angiogenesis, and the vascular response to stimuli.

Applications of SV40 Large T Antigen-Immortalized Cells

Cancer Research

Immortalized cells are used to study the mechanisms of tumorigenesis, particularly how cells evade senescence and become cancerous. SV40 Large T antigen plays a role in mimicking early oncogenic events.

Virology

SV40 Large T antigen-immortalized cell lines are widely used for studying viral replication, host-virus interactions, and screening antiviral compounds.

Drug Discovery and Toxicology

These cells provide a consistent and reproducible model for high-throughput drug screening and toxicology studies.

Molecular Biology

Immortalized cell lines are invaluable for molecular biology research, including gene expression studies, protein production, and CRISPR/Cas9 gene editing.

Stem Cell and Regenerative Medicine

In some cases, immortalized cell lines can be used to understand the differentiation potential of stem cells or to model cellular processes in regenerative medicine.

Considerations

Altered Cellular Functions: Due to the inhibition of p53 and Rb, cells immortalized with SV40 Large T antigen may exhibit changes in their behavior or differentiation capacity.

Monitoring Genetic Stability: Regularly monitor the genetic stability of SV40 Large T antigen-immortalized cells, as they may acquire mutations over time.

In Vivo Applications: Caution should be taken when using these cells for in vivo applications, as they may have an increased risk of tumorigenicity.

In summary, SV40 Large T antigen is a powerful tool for immortalizing a wide variety of cell types, enabling them to proliferate indefinitely. While it is highly efficient and widely used, careful consideration of the effects on cellular behavior and genetic stability is necessary to ensure valid experimental results.

1012 月/24

KMD Bioscience-FACS Sorting Protocol

2024-12-10CRO ServiceFACS Sorting Protocolkmdbioscience

Fluorescence-activated cell sorting (FACS) is a powerful technique to separate and purify cell populations based on fluorescent labeling. This method uses flow cytometry to sort individual cells based on their specific surface markers, intracellular proteins, or other characteristics like size and granularity. Below is a general FACS sorting protocol that outlines the key steps involved in preparing cells, staining them with fluorescent markers, and sorting them using a flow cytometer equipped with sorting capabilities.

Materials

Cells of interest (e.g., primary cells or cell lines)

Fluorophore-conjugated antibodies (specific to the cell surface or intracellular markers)

Flow cytometry buffer (e.g., PBS with 1-2% FBS or BSA)

Cell strainer (40 µm)

Propidium iodide (PI) or 7-AAD for dead cell exclusion (optional)

DNAse I (optional, for single-cell suspension)

EDTA (optional, to prevent clumping)

Sorting flow cytometer (e.g., FACSAria, MoFlo)

Optional

Fixative (e.g., 1% paraformaldehyde) for intracellular staining

Perm/Wash buffer (e.g., saponin or Triton X-100) for intracellular staining

Blocking reagent (e.g., Fc receptor block, 10% normal serum)

Step-by-Step FACS Sorting Protocol

Prepare Single-Cell Suspension

Primary Cells: If using tissues, dissociate the tissue into a single-cell suspension using enzymatic digestion (e.g., collagenase for solid tissues) or mechanical disruption (e.g., mincing or using a tissue dissociator).

Cell Lines: For adherent cells, detach them using trypsin or an enzyme-free dissociation buffer. Collect cells in flow cytometry buffer (e.g., PBS with 1-2% FBS or BSA).

Filter the Cells: Pass the cell suspension through a 40 µm cell strainer to remove clumps and debris. This is critical to prevent clogging of the flow cytometer.

Optional: Add DNAse I (50-100 µg/mL) if working with tissues to prevent cell clumping caused by free DNA released from dead cells.

Optional: Add 2 mM EDTA to the flow buffer to reduce cell aggregation, particularly when working with sticky cells (e.g., immune cells).

Cell Counting and Viability Check

Count the Cells: Use a hemocytometer or automated cell counter to determine cell concentration.

Check Viability: Perform a viability check using Trypan blue, or for live/dead staining, use propidium iodide (PI) or 7-AAD for dead cell exclusion.

Block Non-Specific Binding (Optional)

Fc Receptor Block: If working with immune cells (e.g., from mouse spleen or blood), block Fc receptors to reduce non-specific binding of antibodies. Use Fc receptor blocking reagent or normal serum from the same species as the fluorophore-conjugated antibodies (e.g., mouse serum for mouse antibodies).

Stain Cells with Fluorophore-Conjugated Antibodies

Surface Markers

Add the fluorophore-conjugated antibodies (e.g., PE, FITC, APC, or BV421) specific to the surface markers of interest (e.g., CD4, CD8, CD19, etc.).

Antibody Concentration: Follow the manufacturer’s recommended concentration (typically 0.5-2 µg/1 million cells).

Incubate the cells with the antibodies on ice or at 4°C for 20-30 minutes in the dark (to protect fluorophores from light).

Wash: After incubation, wash the cells 2-3 times with flow cytometry buffer (PBS + 1-2% FBS/BSA) to remove unbound antibodies. Centrifuge at 300 x g for 5 minutes and discard the supernatant between washes.

Intracellular Staining (If Needed)

If sorting based on intracellular markers (e.g., transcription factors, cytokines), fix and permeabilize the cells after surface staining using a fixation/permeabilization kit or 1% paraformaldehyde and a permeabilization buffer (e.g., saponin or Triton X-100).

Add the fluorophore-conjugated antibody specific to the intracellular marker, and incubate for 30 minutes in the dark at 4°C.

Wash the cells 2-3 times with permeabilization buffer, followed by a wash with flow cytometry buffer.

Dead Cell Exclusion (Optional)

To exclude dead cells from the analysis, add a viability dye such as propidium iodide (PI), 7-AAD, or Zombie Aqua before sorting.

Incubate for 5-10 minutes at room temperature in the dark.

Prepare Cells for Sorting

Resuspend Cells: After the final wash, resuspend the cells in flow cytometry buffer at an appropriate concentration (typically 1 x 10⁶ cells/mL) for FACS sorting.

Filter Again: Pass the cell suspension through a 40 µm cell strainer again to ensure a single-cell suspension and prevent clogging during sorting.

Set Up the Flow Cytometer

Calibrate the Cytometer: Perform calibration using flow cytometry calibration beads or standard beads to set appropriate voltage and sensitivity for each fluorophore.

Gating: Define the gates based on negative controls and single-stained controls. Make sure to set up compensation controls if using multiple fluorophores to account for spectral overlap.

Live-Dead Gating: If a viability dye was used, gate out dead cells based on PI or 7-AAD staining to ensure that only live cells are sorted.

Doublet Discrimination: Use forward scatter (FSC) and side scatter (SSC) to exclude doublets and ensure the sorting of single cells.

Set Sort Criteria: Define sorting gates for the populations of interest based on fluorescence intensity.

Sort the Cells

Sorting Mode: Set the flow cytometer to the appropriate sorting mode (e.g., “Purity” mode for maximum purity, or “Yield” mode for higher numbers of sorted cells but lower purity).

Collect Sorted Cells: Sorted cells can be collected into collection tubes (e.g., 15 mL conical tubes or microtubes) containing collection medium (e.g., RPMI with 10% FBS or PBS with 1-2% BSA) to ensure cell viability after sorting.

Sort Rate: Adjust the sort rate based on the sample concentration and the required purity/yield. High sort rates can reduce purity, so aim for a reasonable balance between speed and purity.

Post-Sort Analysis and Recovery

Post-Sort Purity Check: After sorting, reanalyze a small sample of the sorted cells to confirm the purity of the sorted population.

Cell Recovery: If cells are intended for downstream applications (e.g., RNA extraction, culture, or functional assays), ensure they are placed in appropriate growth medium or lysis buffer immediately after sorting to preserve their viability and functionality.

Optional Steps

Intracellular Staining Controls: For intracellular targets, include unstained and isotype controls to confirm specific staining and rule out background signal.

Fixation of Sorted Cells: If cells need to be fixed post-sorting for downstream analysis (e.g., microscopy), use 1-4% paraformaldehyde to fix the cells.

Key Considerations

Cell Viability: Maintain the cells on ice during the staining and sorting procedures to prevent loss of viability, especially for delicate primary cells.

Fluorophore Selection: Choose fluorophores with minimal spectral overlap and ensure proper compensation controls are used to avoid incorrect gating.

Doublet Exclusion: Use both FSC and SSC to exclude cell doublets during sorting, as they can skew the results and reduce the purity of the sorted populations.

Sorting Pressure: Adjust the sorting pressure on the cytometer based on cell size to avoid damaging the cells.

Applications of FACS Sorting

Stem Cell Research: FACS sorting is used to isolate specific stem cell populations based on surface markers (e.g., CD34+ hematopoietic stem cells).

Immunology: It is used to sort specific immune cell populations (e.g., CD4+ or CD8+ T cells, B cells, dendritic cells) for downstream functional assays, such as cytokine production or proliferation.

Cancer Research: Tumor cells can be sorted based on markers like EpCAM or HER2 to study cancer progression or drug responses.

Single-Cell RNA Sequencing: FACS is often used to purify specific cell types before performing single-cell RNA sequencing for transcriptomic analysis.

FACS sorting is a powerful technique for isolating specific cell populations based on defined markers. By following this protocol, researchers can obtain highly pure and viable cell populations for downstream applications, including cell culture, molecular analysis, and functional assays.

0712 月/24

KMD Bioscience-Primary vs Immortalized Cells

2024-12-07CRO ServicePrimary vs Immortalized Cellskmdbioscience

Primary cells and immortalized cells are two distinct types of cells used in biological and biomedical research. Each type has its own advantages and limitations, depending on the research goals. Below is a comparison of primary cells and immortalized cells in various aspects, including their characteristics, advantages, and disadvantages.

Definition

Primary Cells

These are cells isolated directly from tissues or organisms (e.g., human, mouse) and cultured in vitro. Primary cells closely resemble the physiology and behavior of cells in vivo and retain the specific characteristics of the tissue or organ from which they are derived.

Immortalized Cells

Immortalized cells are cells that have been genetically modified (or have undergone spontaneous mutations) to proliferate indefinitely. They bypass normal cellular senescence, allowing for continuous cell division and long-term culture. Immortalization is typically achieved by introducing oncogenes, viral proteins (e.g., SV40 Large T antigen, HPV E6/E7), or by overexpressing hTERT (human telomerase reverse transcriptase).

Lifespan and Growth Characteristics

Primary Cells

Finite Lifespan: Primary cells have a limited proliferative capacity. They can only undergo a certain number of divisions before entering senescence, at which point they stop dividing.

Slower Growth: They tend to grow more slowly than immortalized cells because they reflect normal, differentiated behavior. They require optimized culture conditions to maintain their viability.

Morphological Stability: Primary cells generally maintain the morphology and functional characteristics of the tissue from which they were derived.

Immortalized Cells

Indefinite Lifespan: Immortalized cells can divide indefinitely and do not undergo senescence, making them ideal for long-term experiments.

Faster Growth: Immortalized cells typically grow faster than primary cells and can reach confluency more quickly in culture. They often require less specific conditions compared to primary cells.

Genetic Instability: Immortalized cells can accumulate mutations over time, which can lead to changes in cell behavior and phenotype.

Physiological Relevance

Primary Cells

High Physiological Relevance: Primary cells closely mimic the in vivo behavior, gene expression, and function of the tissue they were derived from. They are often more biologically relevant for studying physiological processes, drug effects, and disease mechanisms.

Natural Gene Expression: Gene expression in primary cells reflects their natural environment, making them more suitable for studying gene regulation, signaling pathways, and responses to stimuli.

Immortalized Cells

Reduced Physiological Relevance: Immortalized cells often differ from their original tissue in terms of gene expression and behavior due to the genetic modifications used to bypass senescence. This makes them less reflective of in vivo conditions.

Altered Phenotype: Some immortalized cells may lose key functional properties of the original cell type, particularly if they accumulate genetic changes over time.

Experimental Use and Applications

Primary Cells

Drug Testing and Toxicology: Primary cells are used in preclinical testing to assess the efficacy and toxicity of drugs, as they provide a more accurate reflection of how human tissues may respond.

Disease Modeling: They are often used for studying specific disease mechanisms in a more physiologically relevant setting, such as cancer, cardiovascular diseases, and neurodegenerative disorders.

Personalized Medicine: Primary cells can be derived from patients, enabling personalized approaches to study disease progression and drug responses in the context of the patient’s own genetic background.

Immortalized Cells

High-Throughput Screening: Immortalized cells are widely used in high-throughput drug screening and biotechnology applications due to their rapid growth and ease of culture. They are used in large-scale assays to identify potential therapeutic targets.

Gene Editing and Transfection: These cells are easier to genetically manipulate (e.g., via CRISPR/Cas9 or plasmid transfection), making them useful for studies involving gene function, protein production, and pathway analysis.

Long-Term Experiments: Due to their indefinite lifespan, immortalized cells are ideal for experiments requiring long-term cell culture, such as cancer research and cell signaling studies.

Advantages and Disadvantages

Primary Cells

Advantages

More Physiologically Relevant: They maintain the characteristics and function of the tissue they are derived from.

Accurate Disease Models: They are more likely to represent the natural cellular environment, making them useful for disease modeling and testing.

Diverse Applications: Primary cells from various tissues (e.g., neurons, hepatocytes, fibroblasts) can be used to study tissue-specific functions.

Disadvantages

Limited Lifespan: Cells undergo senescence after a finite number of divisions, limiting their long-term use.

Difficult to Culture: Primary cells often require optimized and complex culture conditions. They are more sensitive to environmental changes and may need special growth factors and media.

Heterogeneity: Primary cells from different individuals may behave differently due to genetic variability, making reproducibility a challenge in some cases.

Immortalized Cells

Advantages

Unlimited Proliferation: They can be cultured indefinitely, which is advantageous for long-term experiments and large-scale studies.

Easier to Culture: Immortalized cells generally require less stringent growth conditions and are less sensitive to culture changes.

Genetic Manipulation: They are easier to transfect or genetically manipulate, making them valuable for research into gene function and protein production.

Reproducibility: Because immortalized cell lines are clonal and can be easily shared between labs, they offer more experimental consistency and reproducibility.

Disadvantages

Reduced Physiological Relevance: Genetic modifications can lead to changes in cell behavior, reducing their ability to mimic in vivo conditions.

Accumulation of Mutations: Immortalized cells may acquire mutations over time, potentially leading to altered cell function, and even transformation into cancer-like cells.

Altered Functionality: Some immortalized cells may lose differentiation potential or other specific functions, making them less suitable for certain studies.

Examples

Primary Cells

Human Dermal Fibroblasts (HDFs): Used for studying wound healing, fibrosis, and aging.

Primary Neurons: Used in neuroscience research to study neurodegenerative diseases and neural signaling.

Primary Hepatocytes: Utilized in drug metabolism and toxicology studies.

Immortalized Cells

HEK 293T (Human Embryonic Kidney): Widely used for protein production and gene editing studies.

HeLa Cells: One of the oldest and most widely used cancer cell lines, used in cancer biology and virology research.

MCF-7 (Breast Cancer Cells): Used to study breast cancer mechanisms and drug responses.

Summary of Primary vs. Immortalized Cells

Feature	Primary Cells	Immortalized Cells
Lifespan	Finite, limited by senescence	Infinite, can divide indefinitely
Physiological Relevance	High, closely mimic in vivo cells	Lower, may differ from in vivo
Growth Rate	Slow	Fast
Ease of Culture	More difficult, specialized media	Easier, less stringent conditions
Reproducibility	Variable due to donor differences	High due to clonal populations
Genetic Stability	Stable but prone to senescence	Prone to genetic changes over time
Applications	Drug testing, disease modeling	High-throughput screening, gene editing
Example	Primary hepatocytes, neurons	HEK 293T, HeLa cells

Both primary and immortalized cells have distinct advantages and limitations, and the choice between them depends on the specific requirements of the experiment. Primary cells are preferable for experiments that require physiological relevance, while immortalized cells are better suited for experiments that require long-term culture, reproducibility, and ease of manipulation.

0612 月/24

KMD Bioscience-Cell Immortalization Protocol

2024-12-06CRO ServiceCell Immortalization Protocolkmdbioscience

A cell immortalization protocol is a detailed set of instructions used to make primary cells proliferate indefinitely, bypassing their natural limit on division (senescence). The method used to immortalize cells can vary based on the specific technique (e.g., hTERT overexpression, SV40 Large T antigen, HPV E6/E7, etc.) and the type of cells being immortalized. Below is a general protocol for one of the most common methods: immortalization using human telomerase reverse transcriptase (hTERT).

Protocol for hTERT-Mediated Cell Immortalization

Materials

Primary cells (e.g., fibroblasts, epithelial cells)

Lentiviral vector containing hTERT (commercially available or self-constructed)

Lentiviral packaging plasmids (for lentivirus production)

Lipofectamine or other transfection reagent

HEK 293T cells (for producing lentiviral particles)

Polybrene (to enhance transduction efficiency)

Puromycin or another selection antibiotic (if using a selectable marker)

Dulbecco’s Modified Eagle Medium (DMEM) or another appropriate growth medium for primary cells

Fetal bovine serum (FBS)

Phosphate-buffered saline (PBS)

Trypsin-EDTA solution

Incubator set to 37°C with 5% CO₂

Day 1: Prepare Lentiviral Particles (if not purchasing pre-made)

Transfection of HEK 293T Cells:

Seed HEK 293T cells in a 10 cm dish at approximately 70-80% confluency.

Prepare a transfection mix containing the lentiviral packaging plasmids, the lentiviral vector encoding hTERT, and a transfection reagent like Lipofectamine in serum-free DMEM.

Add the transfection mix dropwise to the HEK 293T cells and incubate for 6-8 hours.

After incubation, replace the medium with fresh, complete DMEM containing 10% FBS.

Collect Lentiviral Supernatant:

After 48-72 hours, collect the lentiviral-containing supernatant from the transfected HEK 293T cells.

Filter the supernatant using a 0.45 µm syringe filter to remove cell debris.

Optional: Concentrate the lentivirus using ultracentrifugation or commercially available viral concentration kits, if desired.

Day 3: Transduce Primary Cells with hTERT Lentivirus

Prepare Primary Cells:

Seed the primary cells (e.g., fibroblasts, epithelial cells) in a 6-well plate at 50-70% confluency in complete growth medium.

Lentiviral Transduction:

Add 2-10 µg/mL of polybrene (a cationic polymer that enhances viral transduction) to the culture medium.

Add the filtered hTERT-containing lentiviral supernatant to the cells at an appropriate multiplicity of infection (MOI) based on the cell type.

Incubate the cells with the viral supernatant for 24 hours in a humidified incubator at 37°C and 5% CO₂.

Medium Replacement:

After 24 hours, replace the viral supernatant with fresh, complete medium and continue incubation for an additional 48-72 hours.

Day 6: Selection of Transduced Cells (Optional)

Antibiotic Selection:

If the lentiviral vector contains an antibiotic resistance marker (e.g., puromycin or neomycin), begin selecting the successfully transduced cells by adding the corresponding antibiotic to the growth medium.

Use puromycin at a concentration of 1-2 µg/mL for 3-5 days, depending on the sensitivity of your cell type.

Continue antibiotic selection until all non-transduced (uninfected) cells die, and only the antibiotic-resistant, hTERT-transduced cells survive.

Expand Surviving Cells:

Once antibiotic selection is complete, allow the surviving hTERT-transduced cells to expand in fresh medium. Change the medium every 2-3 days until the cells reach 70-80% confluency.

Day 10+: Validation and Expansion

Confirm hTERT Expression:

Validate the immortalization by confirming the expression of hTERT in the transduced cells using quantitative PCR (qPCR) or Western blotting.

Assess telomerase activity using the Telomerase Repeat Amplification Protocol (TRAP) assay, if desired.

Test for Proliferation and Senescence:

Perform proliferation assays (e.g., cell counting or MTT assays) to confirm that the hTERT-immortalized cells have extended their proliferative capacity compared to control (non-immortalized) cells.

Stain the cells for β-galactosidase, a marker of senescence, to verify that the immortalized cells are not senescent.

Expand and Freeze:

Once immortalization is confirmed, expand the hTERT-immortalized cells and cryopreserve them in freezing medium (e.g., 10% DMSO in FBS) for future experiments.

Notes and Considerations

Primary Cell Source: The success of immortalization depends on the source and type of primary cells. Some cells (e.g., fibroblasts) are easier to immortalize than others (e.g., neurons or myocytes).

Antibiotic Selection: If you use an antibiotic-resistant marker, optimize the concentration of the antibiotic for your cell type. Non-optimized concentrations can lead to either incomplete selection or cell death.

Verification: Always verify that the immortalized cells retain their original characteristics (e.g., morphology, differentiation potential) after immortalization, as some immortalization methods can alter cell behavior.

Alternative Immortalization Methods

If you are using SV40 Large T Antigen or HPV E6/E7, the procedure remains similar, but instead of introducing hTERT, you will use vectors that express SV40 large T antigen or HPV E6/E7 proteins. The following modifications apply:

Viral Vectors: Use plasmids or viral vectors carrying the SV40 large T antigen or HPV E6/E7 genes instead of hTERT.

Confirmation: After transduction, confirm immortalization by checking for the expression of SV40 T antigen or HPV proteins using PCR or Western blotting.

Final Thoughts

Immortalization allows for the generation of stable cell lines that can proliferate indefinitely, facilitating long-term studies in areas like cancer biology, drug screening, and tissue engineering. However, some immortalization methods can introduce genetic or functional changes, so validation and comparison with primary cells are important for accurate experimental outcomes.

0512 月/24

KMD Bioscience-Cell Immortalization Techniques

2024-12-05CRO ServiceCell Immortalization Techniqueskmdbioscience

Cell immortalization is the process by which primary cells, which normally have a finite lifespan, are modified to proliferate indefinitely. Several techniques are used to immortalize cells, depending on the cell type and the desired characteristics of the immortalized line. Below are some of the common techniques used for cell immortalization:

Telomerase Overexpression (hTERT Immortalization)

Mechanism: Telomerase is an enzyme that maintains the length of telomeres, the protective caps at the ends of chromosomes. In most somatic cells, telomeres shorten with each cell division, eventually leading to cellular senescence or death. Overexpressing the human telomerase reverse transcriptase (hTERT) gene in cells can prevent telomere shortening, thereby allowing the cells to bypass senescence and continue dividing indefinitely.

Advantages: This method closely mimics natural mechanisms of cellular immortalization (as seen in stem cells and cancer cells). It often preserves the normal functions and characteristics of the cells.

Applications: This method is widely used to immortalize primary human cells, such as fibroblasts and epithelial cells.

SV40 Large T Antigen (SV40 LT)

Mechanism: The SV40 large T antigen is a viral protein derived from the Simian Virus 40 (SV40). It interferes with the function of tumor suppressor proteins such as p53 and retinoblastoma protein (pRb), which are key regulators of the cell cycle. By inactivating these proteins, the SV40 large T antigen drives cells into continuous proliferation and immortalization.

Advantages: This is a widely used technique that is effective for a variety of cell types, including epithelial cells, fibroblasts, and endothelial cells.

Disadvantages: Cells immortalized with SV40 large T antigen may undergo additional genetic changes over time, which can alter their normal phenotype.

Human Papillomavirus (HPV) E6/E7 Oncoproteins

Mechanism: E6 and E7 are viral oncoproteins derived from the Human Papillomavirus (HPV). The E6 protein binds to and degrades the tumor suppressor protein p53, while E7 binds and inactivates the retinoblastoma (pRb) protein. Together, these oncoproteins disrupt normal cell cycle regulation, leading to continuous cell proliferation and immortalization.

Advantages: Effective for immortalizing epithelial cells, especially human keratinocytes and other cell types closely associated with HPV-related pathologies (e.g., cervical cells).

Disadvantages: As with SV40, the use of viral oncoproteins can lead to additional genetic changes that may affect the cells’ behavior over time.

Epstein-Barr Virus (EBV)

Mechanism: EBV is a herpesvirus that can immortalize B-lymphocytes by expressing the EBV nuclear antigen 2 (EBNA-2) and latent membrane protein 1 (LMP-1). These proteins drive cell proliferation and prevent apoptosis, leading to the continuous growth of B cells.

Applications: This method is specifically used to immortalize B cells and generate lymphoblastoid cell lines, which are valuable for studying immune responses and genetic diseases.

Advantages: EBV-immortalized B cells maintain many of the properties of primary B lymphocytes.

KRAS or MYC Oncogene Overexpression

Mechanism: Overexpression of oncogenes such as KRAS or MYC can drive uncontrolled cell proliferation and immortalization by promoting growth factor-independent signaling and bypassing cell cycle checkpoints.

Advantages: Effective for a variety of cell types, particularly epithelial cells and fibroblasts.

Disadvantages: Oncogene overexpression may alter normal cell function and lead to tumorigenic changes, which could complicate experiments.

CRISPR/Cas9-Mediated Knockout of Tumor Suppressors

Mechanism: CRISPR/Cas9 gene-editing technology can be used to specifically knock out tumor suppressor genes such as p53 or p16 in cells. By removing these key cell cycle regulators, the cells can bypass senescence and continue proliferating indefinitely.

Advantages: This method allows for precise genetic modifications and can be tailored to specific cells or experimental needs.

Disadvantages: Knockout of tumor suppressors may lead to unwanted changes in cell physiology over time.

Spontaneous Immortalization

Mechanism: In some cases, cells can undergo spontaneous immortalization after prolonged culture or due to inherent genetic instability. This often occurs in mouse fibroblasts (e.g., NIH 3T3 cells), but it is less common in human cells.

Disadvantages: Spontaneous immortalization is unpredictable and often leads to significant genetic changes in the cells.

Hybridoma Technology (Specific to B Cells)

Mechanism: Hybridomas are created by fusing a normal antibody-producing B cell with an immortal myeloma cell line. The resulting hybrid cell line can proliferate indefinitely and produce monoclonal antibodies specific to the original B cell’s antigen.

Applications: This technique is widely used for producing monoclonal antibodies for research, diagnostics, and therapeutic purposes.

Considerations for Immortalization

Cell Type Specificity: Different immortalization techniques work better with certain cell types. For example, hTERT is often used for fibroblasts and epithelial cells, while EBV is specific to B cells.

Alteration of Normal Function: Immortalized cells may not always perfectly mimic the behavior of primary cells, especially if viral or oncogenic methods are used. It’s important to validate the functionality of the immortalized cells for the intended application.

Risk of Tumorigenicity: Immortalization, particularly through viral oncogenes, can increase the likelihood of tumorigenic changes, so caution should be taken when using these cells for certain studies, especially in vivo experiments.

These techniques are widely employed in research and biomedicine, helping to create valuable cell lines for studying cancer biology, drug development, and gene therapy.

2911 月/24

KMD Bioscience-T Cell Immortalization

2024-11-29CRO Servicet cell immortalizationkmdbioscience

Immortalizing T cells is a complex process due to their highly specialized and differentiated nature. Immortalization is used to generate long-term T cell lines that retain their antigen-specific properties, allowing for continuous studies on T cell biology, immunotherapy, and drug development. Several methods have been developed for T cell immortalization, including the use of viral oncogenes, telomerase overexpression, and hybridoma technology (for mouse T cells). Below are the most common methods and protocols used to immortalize T cells.

This protocol focuses on a combination of hTERT overexpression and SV40 Large T antigen, which is commonly used to immortalize human T cells while maintaining their functional properties.

Materials

Primary T cells (isolated from peripheral blood mononuclear cells (PBMCs) or lymph nodes)

Lentiviral vector containing hTERT or SV40 Large T antigen

Lentiviral packaging plasmids (if generating your own virus)

Transfection reagent (e.g., Lipofectamine 2000)

Polybrene (for enhancing transduction efficiency)

Selection antibiotic (e.g., puromycin)

RPMI-1640 medium (or other suitable T cell medium)

T cell growth factors (e.g., IL-2, IL-7, IL-15)

Incubator set to 37°C with 5% CO₂

Step-by-Step Protocol

Day 1: Virus Production (if generating lentiviral particles)

HEK 293T Cell Transfection:

Seed HEK 293T cells at 70-80% confluency in a 10 cm dish.

Transfect the HEK 293T cells with the hTERT or SV40 Large T antigen plasmid along with packaging plasmids using a transfection reagent (e.g., Lipofectamine 2000 or PEI).

Incubate for 48-72 hours.

Collect Lentiviral Supernatant:

Harvest the virus-containing supernatant and filter it through a 0.45 µm filter to remove cell debris.

Concentrate the virus using ultracentrifugation or a commercial viral concentration kit (optional).

Day 3: Transduction of Primary T Cells

Prepare Primary T Cells:

Isolate T cells from PBMCs using density gradient centrifugation or magnetic bead separation (e.g., CD3+ T cell isolation kit).

Seed the T cells in a 6-well plate in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin, and IL-2 (50-100 IU/mL).

Lentiviral Transduction:

Add polybrene (5-10 µg/mL) to the medium to enhance transduction efficiency.

Add the concentrated lentiviral supernatant containing hTERT or SV40 Large T antigen to the T cells.

Incubate for 24 hours at 37°C.

Medium Replacement:

After 24 hours, replace the lentiviral supernatant with fresh medium containing IL-2 (50-100 IU/mL) and continue to culture the cells.

Day 6: Selection of Transduced Cells (Optional)

Antibiotic Selection:

If the lentiviral vector contains an antibiotic resistance gene (e.g., puromycin), begin selection by adding the appropriate antibiotic to the culture medium (e.g., puromycin at 1-2 µg/mL).

Select for several days until non-transduced cells are eliminated.

Expand Surviving Cells:

Expand the surviving immortalized T cells in medium supplemented with IL-2, IL-7, or IL-15 to promote T cell growth.

Validation of Immortalization

Check for hTERT or SV40 Large T Antigen Expression:

Validate the expression of hTERT or SV40 Large T antigen using Western blot, qPCR, or immunofluorescence.

Check for telomerase activity using the TRAP assay (Telomeric Repeat Amplification Protocol) if using hTERT.

Proliferation Assay:

Monitor the growth rate of the immortalized T cells to confirm extended proliferative capacity compared to non-immortalized controls.

Functional Assays:

Perform functional assays such as TCR stimulation, cytokine secretion (e.g., IFN-γ, IL-2), and cytotoxicity assays to confirm that the T cells retain their immune functions.

Senescence Assay:

Perform β-galactosidase staining to ensure that the immortalized T cells are not undergoing senescence.

Applications of Immortalized T Cells

Immunotherapy:

Immortalized T cells are used to study the role of T cells in cancer immunotherapy, including CAR-T cell therapies.

T Cell Receptor (TCR) Research:

Immortalized T cells can be used to explore TCR signaling, antigen specificity, and T cell activation in response to pathogens or tumors.

Autoimmune Disease Studies:

Immortalized T cell lines are used to study the role of autoreactive T cells in diseases like multiple sclerosis, type 1 diabetes, and rheumatoid arthritis.

Infectious Disease Research:

These cells are employed to investigate T cell responses to viral, bacterial, and parasitic infections.

Conclusion

Immortalizing T cells is a powerful tool for generating stable and long-lasting T cell lines. Methods such as hTERT overexpression, SV40 Large T antigen, or oncogene-driven immortalization allow researchers to conduct long-term studies on T cell function, signaling, and immunotherapy. However, it is important to validate that the immortalized cells retain their functional properties and do not undergo unwanted transformation.