Types of Growth Factors for Common Cell Types

Growth and attachment factors, natural and recombinant, are used in conjunction with other essential nutrients and hormones to maximize biological stimulation. The optimal conditions and concentrations of growth factors for cell types must be empirically determined. The 5 main types of growth factors used in cell culture are:

• EGF (epidermal growth factor), used for culturing epithelial and fibroblast cells
• FGF-2 (basic fibroblast growth factor), also known as FGF basic or bFGF and used for maintenance of undifferentiated cells
• BDNF (brain-derived neurotrophic factor), for promoting neuronal survival and differentiation
• VEGF (vascular endothelial growth factor), used to induce endothelial cell proliferation and angiogenesis
• GM-CSF (granulocyte macrophage colony-stimulating factor), for modulating hematopoietic cells

The following table provides a summary of conditions for growth and attachment factor supplementation reported to give maximal stimulation for various cell lines. It can be used to establish initial concentrations prior to optimization; for the best results please refer to your product’s growth factor specifications.

The following list of materials is generic and should be used as a guide to collect the necessary materials to complete your experiment. For a complete list of materials needed, please refer to the product-specific instructions for your cell line.

• Laminar flow hood
• 70% ethanol
• Personal protective equipment (PPE)
• Vial(s) of your selected cell line
• The appropriate growth supplements, serum, and basal medium to make your desired complete growth medium
• Antibiotics such as Penicillin-Streptomycin (optional)
• Cell culture plates, dishes, and flasks
• Sterile pipettes and pipette tips
• Sterile conical tubes

The following protocol does not specify the appropriate concentration of working medium, serum, reconstitution buffer, or types of growth factors needed to mix a complete growth medium. For specific instructions related to media preparation for your chosen cell lines, download the Product Sheet and Certificate of Analysis (CoA) to obtain the necessary information before beginning your experiment. Failure to do so will result in incorrect concentration or possible damage to the products. Always work in aseptic conditions to avoid contamination.

1. Without opening the vial containing the lyophilized growth factor, start by centrifuging the vial at approximately 13,000 rpm for 30–60 seconds.
2. Reconstitute the growth factor by dissolving the lyophilized protein in the appropriate solvent and concentration according to the lot-specific CoA. Do not add carrier to the recombinant protein’s initial reconstitution solution unless otherwise specified on the CoA. (Most proteins can be reconstituted with the addition of sterile distilled water at a concentration of 0.1 to 1.0 mg/mL. For example, for 100 µg of protein, the amount of water that should be added should be between 100 µL and 1 mL, resulting in a protein solution with a concentration between 1 mg/mL and 0.1 mg/mL. However, the product data sheet or COA will indicate when a diluent other than water is required. Recommended solutions, carrier protein concentrations, and extended storage conditions can also be found within these documents.)
3. Mix carefully by gently pipetting up and down or put the vial cap back on and invert the vial gently a few times. Do not vortex after reconstitution.
4. Spin down the vial for a few seconds.
5. Make storage preparations for your proteins:
   • The reconstituted protein solution can be stored at 2–8°C for up to one week until you are prepared to use it in your experiment.
   • To create aliquots for long-term freezer storage, dilute to working concentration using a buffer or medium containing a carrier (0.1% BSA, 5% HSA, 10% FCS, 5% trehalose 2–8˚C –20°C to –80°C.) Distribute to aliquots and store at –20 to –80°C (for 3–12 months, as specified on the CoA.) Aliquots should not have a concentration of less than 1 µg/mL and/or a volume of less than 10 μL.

1. In a sterile laminar flow hood, pipette the required volume of basal growth media into a sterile conical tube or bottle. Ensure all reagents and media are pre-warmed to the appropriate temperature before use.
2. If required, add serum to the basal growth media to achieve the desired final concentration (typically 10% for fetal bovine serum).
3. Mix gently by swirling or inverting the tube/bottle to ensure thorough mixing.
4. Thaw the aliquots of growth factors on ice if they are frozen. Avoid repeated freeze-thaw cycles.
5. Prepare the required concentration of each growth factor according to the specific needs of your cell line. This typically involves diluting the stock solutions to the working concentrations.
6. Using sterile pipettes, add the appropriate volume of each growth factor to the basal media and serum. The final concentrations will depend on the specific requirements of your cell line. See the table above for starting concentrations of growth factors for common cell types. Mix gently by swirling or inverting the tube/bottle to ensure thorough mixing.
7. Label the bottle with the contents and date of preparation.
8. If not used immediately, store the complete growth medium with growth factors at 4°C and use within 1–2 weeks.

1. Reconstitution and Storage Protocol
2. RUO Cytokine CoA
3. Recombinant Proteins Support—Getting Started
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7. Cell Culture Select Tool
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