Adherent and suspension baby hamster kidney cells have a different cytoskeleton and surface receptor repertoire

Abstract

Animal cell culture, with single cells growing in suspension, ideally in a chemically defined environment, is a mainstay of biopharmaceutical production. The synthetic environment lacks exogenous growth factors and usually requires a time-consuming adaptation process to select cell clones that proliferate in suspension to high cell numbers. The molecular mechanisms that facilitate the adaptation and that take place inside the cell are largely unknown. Especially for cell lines that are used for virus antigen production such as baby hamster kidney (BHK) cells, the restriction of virus growth through the evolution of undesired cell characteristics is highly unwanted. The comparison between adherently growing BHK cells and suspension cells with different susceptibility to foot-and-mouth disease virus revealed differences in the expression of cellular receptors such as integrins and heparan sulfates, and in the organization of the actin cytoskeleton. Transcriptome analyses and growth kinetics demonstrated the diversity of BHK cell lines and confirmed the importance of well-characterized parental cell clones and mindful screening to make sure that essential cellular features do not get lost during adaptation.

Fig 1. Expression of integrin αvβ3 and αvβ8 on the cellular surface of the adherently growing cell line BHK179 and two suspension cell lines BHK-InV and BHK-2P.

Cells were stained with antibodies targeting αvβ3 (a) or αvβ8 (b) and were analyzed by flow cytometry. MDBK cells served as positive control for integrin αvβ3, while IB-RS-2 (IBRS) cells served as positive control for the expression of αvβ8. Green dots are used for the respective positive control in each panel. BHK179 cells are presented as black squares, while the suspension cell lines are represented as triangles. The negative control cell line CHO677 is shown as red diamonds. Experiments were performed three times independently. Significance code: **** p < 0.0001.

Fig 2. Expression of heparan sulfate (HS) on the cellular surface of the adherently growing cell line BHK179 and the two suspension cell lines BHK-InV and BHK-2P.

Cells were incubated with an antibody targeting HS and were analyzed by flow cytometry. CHO-K1 cells served as positive control (green circles), while CHO677 cells served as negative control (red diamonds). BHK179 cells are shown as black squares and the suspension cell lines are represented by triangles. Experiments were performed three times independently.

Fig 3. Quantity and distribution of filamentous actin.

The microscopy images give examples of the distribution of filamentous actin in BHK179 cells in adherent growth conditions (a) and in one of the suspension cell lines (BHK-2P) (b). Microscopically, there was no difference between the two suspension cell lines. The median fluorescent intensity (MFI) of filamentous actin staining in BHK-InV and BHK-2P (c) was measured by flow cytometry in two replicates per experiment. The experiment has been performed three times independently. Significance code: **** p < 0.0001.

Fig 4. Transfection efficacy and ability to display proteins on the cellular surface of adherent and suspension cells.

Adherent BHK164 cells and BHK-2P suspension cells were transfected with plasmids expressing EGFP (a) or VSV G protein (b). For transfection, either a low dose (downward arrow) or a high dose (upward arrow) of transfection reagent was used. The percentage of successfully transfected cells was measured with flow cytometry after 24 h incubation at 37 °C. The experiments were performed three times independently. Significance code: *** p < 0.001.

Fig 5. Explorative and differential gene expression analysis between adherent and suspension cell lines.

(a) The principal component analysis (PCA) visualizes the difference in gene expression patterns between the adherent BHK179 cells (yellow triangles) and the two suspension cell lines BHK-InV (grey squares) and BHK-2P (blue circles). (b) The number and fold change of differentially expressed genes for each of the three possible comparisons. Down- and up-regulated genes are indicated in blue and yellow, respectively. Dashed lines indicate the cutoff value of |log2 foldchange| > 1. (c) The Venn diagram summarizes differentially expressed genes when comparing FMDV-susceptible cell lines BHK179 and BHK-InV with the FMDV-resistant cell line BHK-2P. (d) The subset of 553 genes from (c) was used for GO term enrichment analysis focusing on cellular components. (e) Normalized gene counts for different integrin genes in the analyzed cell lines BHK179, BHK-2P and BHK-InV. The integrin genes ITGAV, ITGB1, ITGB3, ITGB6 and ITGB8, corresponding to the integrin subunits αv, β1, β3, β6 and β8, respectively, were analyzed. Significance code: * p < 0.05.