Abstract
Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-l-lysine, poly-l-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-l-lysine and poly-l-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement.
Highlights
In multicellular organism tissues the extracellular space surrounding cells is filled with a complex mixture of macromolecules referred to as the extracellular matrix (ECM)
[6] Integrins are transmembrane proteins in the form of ab heterodimers integral for the ECM protein-cell attachment. This interaction generates a cascade of intracellular signals that can control differential gene expression. [7,8] The signaling response is related to the ECM molecular composition that changes according to the cell response to their micro-environment. [9,10] In this way, the ECM is in constant change to facilitate cell requirements of developmental plasticity
Changes in impedance are translated as the unitless term cell index (CI)
Summary
In multicellular organism tissues the extracellular space surrounding cells is filled with a complex mixture of macromolecules referred to as the extracellular matrix (ECM). The ECM is composed of polysaccharides and proteins, such as laminin, fibronectin, elastin, collagen, and their relative amount is tissue specific. These proteins are embedded in a polysaccharide gel. [6] Integrins are transmembrane proteins in the form of ab heterodimers integral for the ECM protein-cell attachment. This interaction generates a cascade of intracellular signals that can control differential gene expression. Many research groups have been using different ECM proteins in tissue culture to modify cell behavior, primarily cell attachment. Many research groups have been using different ECM proteins in tissue culture to modify cell behavior, primarily cell attachment. [12,13,14,15] in addition to increasing attachment, the coating proteins can affect other aspects of cell biology, influencing the final results of the assay [16]
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