Safer Vaccine Manufacturing – defined, animal-free media for the production of vaccines from diploid cells

Normal human diploid fibroblasts (HDF) cells, WI-38, MRC-5, are accepted cell substrates for viral vaccine production because of their lack of tumorigenic potential. (FDA). Many of the current U.S. vaccines including Hepatitis A, and portions of the MMR II vaccine, are currently manufactured from HDF cells using media supplemented with bovine serum. However, the use of bovine serum negatively impacts the safety profile of these vaccines since serum could potentially contain infectious agents. The best solution is to adopt the use of animal-free media in vaccine production. The use of media with a defined composition is also preferred since definition improves the characterization of the vaccine product and could possibly support improved production consistency.

Development of defined animal-free media was not considered an option for HDF cells in the past due to the requirement for growth factors and hormones. Merten [1] in 2000 described serum-free medium development for vaccine production from HDF cells as “not straightforward because only complex serum-free media supplemented with one or more growth factors can be used” … “development of such media for large scale application is difficult and has not been reported to date”

Let us examine what is known about the medium requirements for HDF cells using WI-38 HDF cells as an example. Christofalo and Phillips initially characterized the requirements for serum-free the growth of Wi-38 cells [2-4]. They found that Wi-38 cells grew at similar rates and achieved similar cell density as serum-supplemented media when grown in a defined medium consisting of MCDB-104 medium supplemented with EGF-1, insulin, and dexamethisone. The requirement for these factors should not be a detriment for use of defined, animal-fee HDF vaccine production media. Recombinant EFG-1 and insulin are available from multiple vendors. Sigma carries recombinant insulin and recombinant EGF, Peprotech has recombinant EGF and InVitria carries Animal-free ITSE, which contains insulin, transferrin, selenium and ethanolamine. Furthermore, use of these components is not atypical as they are also components of other commercial vaccine media (VP-SFM, OptiPro, and SFM4megaVir).

Ham and co-workers [5, 6] confirmed the findings of Cristofalo and further improved the defined HDF medium to produce MCDB 110, derivative of MCDB 104 containing prostaglandins, reducing agents, and defined lipids. Thus, MCDB 110 was specifically developed to support the serum-free growth of diploid fibroblasts in combination with the above factors. Again, these components are currently available in animal-free form from numerous suppliers and are often utilized in serum-free media designed for other cell types.

Walthall and Ham noted that the source of iron is a critical factor for the growth HDF cells [6]. Ferrous sulfate only supported cell growth when added to the medium immediately before use. Kan and Yamane later found that ferrous ion was toxic to HDF cells which was attributed to the oxidation of lipids [7]. Furthermore, they found that ferrous sulfate was quickly oxidized to form an insoluble ferric precipitate. This problem was solved by the use of transferrin, which supplied effective iron delivery without the problem of ferrous sulfate oxidation or cell toxicity (Table). Recombinant transferrin is available from several suppliers including InVitria’s Optiferrin, Sigma’s recombinant transferrin, and Fisher Scientific’s recombinant transferrin.

Response of HDF cells to ferrous sulfate or transferrin in iron-free defined medium (adapted from [7])
Iron SourceNumber of Cells (10₄/Dish)(a)
Aerobic GrowthHypoxic Growth
None1,400 +/- 7001,200 +/- 400
Fe₂SO₄.7H₂ (0.8 mg/L)43,000 +/- 5,00052,000 +/- 2,000
Human Transferrin (Holo)86,000 +/- 4,00093,000 +/- 7,000
a) 1,500 cells/dish plated and counted on day 14.

Yamane and Kan also found that albumin was beneficial for the serum-free growth of HDF cells. The benefit of albumin was confirmed by Ryan et al. who was able to obtain 30 populations doublings from an albumin-supplemented MCDB 110 medium [8]. Affordable recombinant human albumin is available from the following sources InVitria’s Cellastim, Sigma’s recombinant albumin, Fisher Scientific’s recombinant albumin, Sheffield Bioscience’s rAlbumin ACF and Mediatech’s cellgro rhAlbumin for medium development.

In conclusion, there is much evidence to indicate that the use of animal-free media for HDF vaccine cell substrates is feasible. The required hormones and factors for HDF cells have been characterized and are currently available in the market in safe, animal-free form. Furthermore, there are a limited number of commercial fibroblast serum-free media on the market, showing feasibility for the application of animal-free media during vaccine production from HDF substrates.

  1. Merten, O.W., Safety for vaccine(s). 2000. (19003393) Cytotechnology. 34 (3): p. 181-3.
  2. Hosokawa, M., P.D. Phillips, and V.J. Cristofalo, The effect of dexamethasone on epidermal growth factor binding and stimulation of proliferation in young and senescent WI38 cells. 1986. (3011471) Exp Cell Res. 164 (2): p. 408-14.
  3. Phillips, P.D. and V.J. Cristofalo, Classification system based on the functional equivalency of mitogens that regulate WI-38 cell proliferation. 1988. (3360059) Exp Cell Res. 175 (2): p. 396-403.
  4. Phillips, P.D. and V.J. Cristofalo, Growth regulation of WI38 cells in s serum-free medium. 1981. (7023957) Exp Cell Res. 134 (2): p. 297-302.
  5. Bettger, W.J., S.T. Boyce, B.J. Walthall, and R.G. Ham, Rapid clonal growth and serial passage of human diploid fibroblasts in a lipid-enriched synthetic medium supplemented with epidermal growth factor, insulin, and dexamethasone. 1981. (7029539) Proc Natl Acad Sci U S A. 78 (9): p. 5588-92.
  6. Walthall, B.J. and R.G. Ham, Multiplication of human diploid fibroblasts in a synthetic medium supplemented with EGF, insulin, and dexamethasone. 1981. (7023958) Exp Cell Res. 134 (2): p. 303-11.
  7. Kan, M. and I. Yamane, Effects of ferrous iron and transferrin on cell proliferation of human diploid fibroblasts in serum-free culture. 1984. (6706356) In Vitro. 20 (2): p. 89-94.
  8. Ryan, P.A., V.M. Maher, and J.J. McCormick, Modification of MCDB 110 medium to support prolonged growth and consistent high cloning efficiency of diploid human fibroblasts. 1987. (3653260) Exp Cell Res. 172 (2): p. 318-28.