Cellular modulation of polymeric device surfaces: promise of adult stem cells for neuro-prosthetics

Abstract

Minimizing the foreign body response is seen as one critical research strategy for implants especially when designed for immune-privileged organs like the brain. The context of this work is to improve deep brain stimulating devices used in a consistently growing spectrum of psychomotor and psychiatric diseases mainly in form of stiff electrodes. Based on the compliance match hypothesis of biocompatibility we present another step forward using flexible implant materials covered with brain cell-mimicking layers. We covered two types of flexible polyimide films with glandular stem cells derived from pancreatic acini. Using real time-PCR and fluorescent immunocytochemistry we analyzed markers representing various cell types of all three germ layers and stemness. The results demonstrate an unchanged differentiation potential of the polyimide fixated cells as measured by mRNA and protein level. Additionally we developed a fibrinous hydrogel coating to protect them against shear forces upon eventual implantation. By repeating previous analysis and additional metabolism tests for all stages we corroborate the validity of this improvement. Consequently we assume that a stem cell-containing cover may provide a native, fully and actively integrating brain-mimicking interface to the neuropil.

Keywords: fibrin; foreign body response; neural prosthesis; polyimide; stem cell; surface modification.

Figure 1

Cells grown on polyimide (A) and on standard cell culture plastic (B). The light micrograph shows cells as irregular structures on the polyimide films. The horizontal bar shows a boundary between two polyimide pieces (Magnification 6×, Discovery V.8; Carl Zeiss GmbH, Oberkochen, Germany). For normal cell culture plastic the cells are depicted by phase-contrast (Axiovert 40C, Carl Zeiss GmbH, Oberkochen, Germany).

Figure 2

Growth behavior of rat pancreatic stem cells on polyimide°1 (left) and polyimide°2 (right) in passage 9 measured after trypsinization of the samples with the Nucleocounter (ChemoMetec).

Figure 3

Characterization of the mRNA-expression in rat pancreatic stem cells grown on cell culture plastic (A) and polyimide°2 film (B). Cells were analyzed in regard to their transcriptome by utilizing RT-PCR. The subsequent capillary gel electrophoresis shows transcripts typical for all germ layers and stemness.

Figure 4

Immunocytochemical staining of rat pancreatic stem cells grown on cell culture plastic (left column), polyimide°1 from IMM (center column), and polyimide°2 from ISIT (right column). Cells are stained for alpha smooth muscle actin (SMA, first row), glial fibrillary acidic protein (GFAP, second row), nestin (third row), and neurofilaments (fourth row). Secondary antibody in green was FITC-labeled goat-anti-rabbit and in red was Cy3-labeled goat-anti-mouse. The nuclear counterstaining was made with DAPI. All scale bars represent 100 μm. Images are made with an Axioskop2 or Axio Observer.Z1 (both Carl Zeiss GmbH, Oberkochen, Germany).

Figure 5

Effects of shear stress examined by an abrasion experiment. Strong abrasion of the confluent cell layer due to the exerted pulling forces is visible (A). This was prevented by applying the protecting fibrin cover (B). The black dots in the right picture indicate air bubbles in the fibrin gel above the cells. Scale bars represent 100 μm (Axio Observer.Z1, Carl Zeiss GmbH, Oberkochen, Germany).

Figure 6

Immunocytochemical staining of rat pancreatic stem cells for Ki67 grown on polyimide°2 from ISIT. Nuclei were positively stained for Ki67 (A) and showed co-localisation with nuclear counterstain DAPI (B). Scale bars represent 100 μm (Axio Observer.Z1, Carl Zeiss GmbH, Oberkochen, Germany).

Figure 7

Analysis of the transcriptome of rat pancreatic stem cells grown on polyimide°2 film covered with fibrin. The considered markers are assigned below.

Figure 8

Immunocytochemical analysis of rat pancreatic stem cells grown on polyimide°2 film covered with fibrin. Cells are stained for alpha smooth muscle actin (SMA), glial fibrillary acidic protein (GFAP), nestin, and neurofilaments (NF). Secondary antibody in green was FITC-labeled goat-anti-rabbit and in red was Cy3-labeled goat-anti-mouse. The nuclear counterstaining was made with DAPI. Scale bars represent 100 μm (Axio Observer.Z1, Carl Zeiss GmbH, Oberkochen, Germany).

Figure 9

Graph of the comparative dextrose–lactate metabolism of pancreatic stem cells. Dextrose curves for all samples are indicated by a dot at each measurement and lactate curves are indicated by a triangle at each measurement. Dashed lines are used for fibrin samples and solid lines for non-coated samples. Gray and black lines represent the blanks. Green lines are used for samples on cell culture plastic and red lines for samples on polyimide films. Sample names are depicted in the legend.