Exploring the Effect of Culture Medium on Experimental Outcomes

Introduction

Culture medium is often an afterthought in experimental design. While methods, reagents, and equipment have evolved over the decades, many researchers rely on the same culture medium formulations in use since the 1960s.

This case study discusses several considerations for the impact of culture medium selection on experimental outcomes and presents findings from culture media testing for in vitro T cell expansion and function.

Discussion

Cytokine Production in IMDM vs DME/F12
Figure 1. Cytokine production of human macrophages as measured by TNFα and IL-6 when cultured in IMDM and DME/F12.

Culture medium can have a significant impact on several areas of cell cultures.

Metabolism

If a medium lacks certain nutrients, it forces cells to spend energy on synthesis rather than other activities important to an effective culture. Some cells may not survive in extreme conditions when lacking essential amino acids (e.g., phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, histidine). If a medium lacks other non-essential amino acids, cells must also synthesize.

If you aim to measure the production of cytokines, it is critical to give cells the proper nutrients. Figure 1 demonstrates how human macrophages produced 10-fold more TNFα and IL-6 when grown in DME/F12 (a 50:50 mixture of Dulbecco’s Minimal Eagle’s medium and Ham’s F12 medium) compared to IMDM (Iscove’s Modified Dulbecco Medium).

Growth of Mouse Macrophages
Figure 2. Growth of mouse macrophages as measured by relative luminescence when supplemented with either RPMI, RPMI and HT, IMDM, or DME/F12.

Proliferation

Cell division requires an abundance of energy for DNA synthesis and mitosis. Thus, the more nutrients available to the cells, the better the proliferation.

We have found that the addition of HT (hypoxanthine and thymidine) increases proliferation, presumably by providing the building blocks for DNA synthesis rather than relying on the cells’ de novo synthesis.

Figure 2 demonstrates the increased proliferation of mouse bone marrow cells in the presence of M-CSF (Macrophage Colony Stimulating Factor) when supplementing RPMI (Roswell Park Memorial Institute) 1640 medium with HT.

Two media with even more nutrients, IMDM and DME/F12, supported cell growth more so than RPMI 1640 alone, also demonstrated in Figure 2.

Testing the Effects: A Comparison of Culture Media for In Vitro T Cell Expansion and Function

Identifying a reliable culture media to support in vitro T cell studies is an important link in the chain of various immuno-oncology strategies.

We believe it is prudent to identify alternative media that can perform suitably in different conditions and for varied purposes. To address this issue, we have conducted a series of studies comparing the performance of several culture media.

Methods

We compared a variety of culture media (including classic media + supplements as well as new media) to several commercially available T cell media in the generation of primary MLR (mixed-lymphocyte reaction), antigen-recall assay (e.g., CMV, tetanus), antigen-specific T cell proliferation assay, and in anti-CD3/CD28-driven T cell expansion culture.

Results

We found that classical media supplemented with several defined components can support primary in vitro responses as measured by cytokine production. However, sustained T cell proliferation demanded additional supplementation and revealed greater differences between media.

This experiment demonstrates the effect of human AB serum (HS) or fetal bovine serum (FBS) added to the culture medium X-VIVO™ 15 (Lonza, Walkersville, MD). At low peptide concentrations (3 and 10 ng/mL), the presence of HS and FBS inhibits T cell proliferation compared to X-VIVO™ 15 alone, as seen in Figure 3.

Cell growth with addition of peptide antigens
Figure 3. Tetanus toxoid-specific T cells were incubated with APC and the indicated concentrations of peptide antigen. After 4 days incubation at 37°C, the proliferation was measured by addition of CellTiter-Glo®.
Cell Growth in the presence of various media formulations
Figure 4. An MLR conducted in the presence of three media formulations. X-VIVO™ 15 supplemented with human serum and DME/F12 with ITS+Premtx and human albumin supported greater cell growth at low concentrations of stimulating B-LCL.

Conclusion

  1. Supplementation of media with human serum does not support optimal cytokine production in the recall antigen assay.
  2. Minimal supplementation of DME/F12 is sufficient to support cytokine production and proliferation in short-term (5-day) assays. (See Figure 4)
  3. Supplementation of culture medium with serum supports growth but requires higher concentrations of antigen.
  4. Choice of culture medium influences not only growth rate but phenotype and function of resulting T cells.

We are currently developing and evaluating medium for growth of T cells. There is a marked difference in the performance of culture media in the first week of culture when compared to later time points. Several media are capable of supporting T cell growth for the initial culture period, but fail to sustain that growth, which is most likely related to the changes in T cell metabolism that have been observed as T cells develop into memory or effector T cells.

Check back for further study data as we continue to develop culture medium.

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