Growth factors—including cytokines like IL-6, TNF-α, IL-15, IL-7, and INF-γ—are chemical messengers that mediate intracellular communication and regulate cellular functions.1 Although many companies offer a wide array of high-quality cytokines for the in vitro culture of immune cells, stem cells, organoids, and other cell types, finding the right growth factors can be challenging.
Relevant scientific literature is always a great place to start. You will want to research the appropriate growth factors for the signaling pathways and cellular processes under investigation. As an example, researchers performing CAR-T cell work may wish to research which combinations of IL-2, IL-7, IL-15, and IL-21 to use.2 For other cell culture types, growth factors such as DLL4 or VCAM1 would be advised to direct differentiation or limit unwanted cell phenotype growth.
Your selected growth factors should also be compatible with the cell culture system, the media, and other supplements. Papers and protocols from suppliers are often a good starting point to assess compatibility issues. However, the most thorough approach typically involves discussions with the system manufacturer and growth factor suppliers. Many suppliers are willing to not only assist with your application but also have research teams dedicated to evaluating their own products.
Growth factors like cytokines are referred to as ancillary or raw materials in regulatory guidance documents. These ancillary components—which also include cell media—contact the cell therapy materials during manufacturing but are not intended to be a part of the final product.3 In the case of immune cell therapy, regulatory bodies like the FDA do not inspect or certify ancillary materials at manufacturing facilities. Therefore, it is up to the suppliers to ensure that good manufacturing practice (GMP) guidelines are followed for all GMP products.4 Similarly, raw material users must perform due diligence when it comes to auditing their suppliers.
Users will want to purchase materials from reputable companies that offer products manufactured and tested in accordance with FDA GMP and international quality management systems standards from the International Organization for Standardization (ISO).4 Regular audits and initial communication between supplier is key when going through regulatory approval.
Overall, GMP-grade products are similar to research use-only (RUO) grade. However, the difference stems from the quality system that GMP-grade products are manufactured under. When developing therapies with the goal of commercialization, GMP-grade materials are the primary choice for many pharmaceutical companies.4 Working with GMP materials from the beginning saves costs by eliminating the need to transition materials used during early discovery. However, RUO products may be the optimal choice for your research if you do not intend to enter the clinical stage or are still in the discovery or development phase.5
Researchers should determine the optimal concentration (or specific activity) of growth factors based on published literature. You will want to ensure that the chosen concentration aligns with the recommended dosage for your specific cell type. In general, the certificate of analysis (also called the CoA) will have a defined specific activity, as most companies will provide specific activities that are calibrated against relevant WHO international standards. This activity can help guide and optimize the concentration of growth factors that need to be added.
Alternatively, many companies express the biological activity of a given cytokine as the ED50 (effective dose), which is the concentration of the cytokine that induces 50% of the maximum response. ED50 values—often expressed in ng/mL—can easily be converted to a specific activity in units/mg.6 While lower ED50 values are better, higher specific activities are desired.
Researchers will want to consider the stability of growth factors under the intended experimental conditions, including temperature, light, and humidity. Most proteins are stored in a lyophilized or freeze-dried state in which water is removed from the product while it is frozen and placed under a vacuum.
Researchers must also follow proper storage guidelines to prevent degradation and be aware of expiration dates. Like most proteins, growth factors are more stable when stored in an undiluted and concentrated form.7 Furthermore, materials should always be reconstituted according to specific instructions found in the certificate of analysis.
Many companies recommend that the lyophilized product be stored at –20°C or lower for long-term storage. Furthermore, growth factors should not be repeatedly frozen and thawed.7 Many certificates of analysis will provide information about long-term stability. However, it is essential to follow your product’s specific instructions.
Many companies offer growth factors that are free of all animal components. Furthermore, products should be certified as being free of transmissible spongiform encephalopathies (TSE) (including bovine spongiform encephalopathies [BSE]).4 High-quality products will also be certified free of microbes, viruses, endotoxins, mycoplasma contamination, and acute/abnormal toxicity.4
The certificate of analysis will also typically provide information about the purity of the growth factors. It is generally recommended that proteins have the highest purity possible (ideally > 95%) to ensure your product is not contaminated with other proteins or endotoxins.5
Many companies will also evaluate the bioactivity of products between lots to investigate batch-to-batch consistency.4 In some cases, the certificate of analysis will also document lot-to-lot variability. Although GMP-manufactured materials are expected to demonstrate consistency, users may want to request certificates of analysis.
More information can be found here.
1. Growth Factors and Cytokines. MilliporeSigma web page. Last accessed: February 2024.
2. Demonstrating How to Enhance Antitumor Activity and Overcome T Cell Exhaustion News Medical: Life Sciences. August 2, 2022.
3. Atouf F, Provost NM, Rosenthal FM. Standards for Ancillary Materials Used in Cell- and Tissue-Based Therapies. BioProcess International. September 1, 2013.
4. GMP Grade Cytokines ACROBiosystems web page. Last accessed: February 2024.
5. How to Choose Your Recombinant Proteins, Cytokines & Growth Factors Proteintech web page. Last accessed: February 2024. /
6. Converting ED50 (ng/mL) to Specific Activity (units/mg). Thermo Fisher Scientific web page. Tech Tip: Specific Activity. 2022. Last accessed: February 2024.
7. Growth Factors for Cell Culture Thermo Fisher Scientific web page. Last accessed: February 2024.