May 1, 2006 (Vol. 26, No. 9)

Maximizing the Success of In Vitro Cell Growth through Novel Surface Coatings

Many factors contribute to the success of in vitro cell growth. Although the characteristics of the cell and the media used play a critical role in this success, surface chemistry (i.e., cell-to-surface interaction) must also be considered.

Typically, adherent cells grow well on polystyrene surfaces that are modified by the addition of oxygen. This changes the surfaces naturally occurring hydrophobic state to a more hydrophilic state that improves cell attachment and growth. However, many primary, transformed, and neuronal cells are fastidious about different surface chemistries. These cells have complex nutritional or other requirements and exhibit weak adherence to traditional cell culture surfaces.

A major focus in the development of surface coatings includes those optimized for fastidious cells. Some of these cells show promise in the discovery and development of vaccines, production of antibodies, and gene therapy. Widely used surfaces for fastidious cell culturing include poly-d-lysine, collagen, and fibronectin; these coatings significantly improve cell attachment and growth.

While greatly improving results with some cell lines, these materials are inherently variable and may be derived from animals, raising the concern of cell culture contamination. These coatings can be considered aseptic but they may not be considered formally sterile, since many do not survive terminal sterilization methods.

In its efforts to develop new and unique surfaces for cell culture, Nalge Nunc International ( developed a non-biological, synthetic analog of poly-d-lysine for plates and dishes. CC3 (CC for cell culture) has been shown to provide a suitable surface for fastidious cell culture in both normal and low-serum media and can be terminally sterilized to a Sterility Assurance Level (SAL) of 10-6. By providing a surface that can be terminally sterilized without affecting its properties, the CC3 surface minimizes contamination at all stages, from research to large-scale production.

CC(3) Labware vs. Poly-d-lysine

The growth of three transformed cell lines (CHO, HEK-293, and BHK) on the CC3 surface was compared to a PDL-coated surface in both normal and reduced serum media. Because neurons often prefer PDL surfaces, the growth of a neuron-like cell line (PC12) was also compared on both PDL and CC3 (with serum only). Stability of the CC3 surface was determined via an accelerated aging study.

All cell lines used in this study were obtained from the American Type Culture Collection (ATCC). Examples of applications for each cell line include the study of cell differentiation and cancer research (PC12), bioproduction of proteins (HEK293), antibody production (CHO), and vaccine production (BHK).

Ninety CC3 plates sterilized to an SAL of 10-6 were produced. The cell lines were seeded onto both the CC3 and poly-d-lysine coated plates in media with serum as recommended by ATCC: HEK293 and CHO, 10&#37 fetal bovine serum medium; BHK, 10&#37 bovine calf serum medium; and, PC12, 15&#37 horse serum medium and 2.5&#37 fetal bovine serum media.

To compare CC3 and poly-d-lysine in reduced serum media, HEK, CHO, and BHK cells were gradually adapted from 10&#37 serum media into reduced serum media (5&#37, 2&#37, and 1&#37) over the course of four passages. The three cell lines were then cultured onto both CC3 and poly-d-lysine coated plates. All plates were then incubated at 37C.

To test stability, CC3 plates were aged in an accelerated program. A total of sixty CC3 plates were aged either two or five years using an aging method in accordance with AAMI/ISO guidelines. Aged plates were compared to one-month old CC3 plates with HEK cells in 10&#37 fetal bovine serum medium.

For analysis, the cells were fixed in 4&#37 formaldehyde and stained in crystal violet to compare the level of cell attachment and growth.

Fig.1: Comparison of CC3 vs. PDL surfaces in normal serum media. HEK and CHO were cultured in 10% fetal bovine serum media, BHK in 10% bovine calf serum media, and PC12 cells in 17.5% serum media.

Results and Discussion

All four cell lines, HEK293, CHO, BHK, and PC12, showed equivalent cell growth when grown at either normal or reduced serum levels (Figure 1).

No decrease in performance was observed between the aged plates and fresh CC3 plates, suggesting the surface will be stable for at least five years at room temperature.


For a group of cells widely used in drug discovery and bioproduction, CC3 demonstrated functional equivalence to poly-d-lysine. It promoted cell attachment and growth in both normal and reduced serum media. Accelerated aging suggests the surface is stable for at least five years at room temperature.

As cell-based assays and bioproduction increase, so too will the need for cell culture surfaces that optimize cell growth under different culture conditions. Creating the CC3 surface expands the options available for the growth of fastidious cell lines&#8212providing a reproducible, stable, and sterile analog of PDL.

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