Dissociated Neurons and Glial Cells Derived from Rat Inferior Colliculi after Digestion with Papain

<div><p>The formation of gliosis around implant electrodes for deep brain stimulation impairs electrode–tissue interaction. Unspecific growth of glial tissue around the electrodes can be hindered by altering physicochemical material properties. However, in vitro screening of neural tissue–material interaction requires an adequate cell culture system. No adequate model for cells dissociated from the inferior colliculus (IC) has been described and was thus the aim of this study. Therefore, IC were isolated from neonatal rats (P3<b>_</b>5) and a dissociated cell culture was established. In screening experiments using four dissociation methods (Neural Tissue Dissociation Kit [NTDK] T, NTDK P; NTDK PN, and a validated protocol for the dissociation of spiral ganglion neurons [SGN]), the optimal media, and seeding densities were identified. Thereafter, a dissociation protocol containing only the proteolytic enzymes of interest (trypsin or papain) was tested. For analysis, cells were fixed and immunolabeled using glial- and neuron-specific antibodies. Adhesion and survival of dissociated neurons and glial cells isolated from the IC were demonstrated in all experimental settings. Hence, preservation of type-specific cytoarchitecture with sufficient neuronal networks only occurred in cultures dissociated with NTDK P, NTDK PN, and fresh prepared papain solution. However, cultures obtained after dissociation with papain, seeded at a density of 2<b>×</b>10⁴ cells/well and cultivated with Neuro Medium for 6 days reliably revealed the highest neuronal yield with excellent cytoarchitecture of neurons and glial cells. The herein described dissociated culture can be utilized as in vitro model to screen interactions between cells of the IC and surface modifications of the electrode.</p></div

Synoptic representation of the proteolytic enzymes used for dissociation.

<p><i>Each assay includes at least two independent preparations with 2–10 wells;</i></p><p><i>w/ = with DNase I.</i></p><p><i>w/o = without DNase I.</i></p

Fluorescence images of different antibodies for glial cell characterization.

<p>Papain digested cells were labelled with GFAP (red) and MAG (green) and are depicted in the first row. Positive staining for GFAP as well as MAG discriminate astrocytes as well as oligodendrocytes, respectively. A second astrocytic marker (S100, red) was also tested in combination with TUJ1 (green) and presented in the second row. Positive staining for S100 confirmed previously obtained results with GFAP (second row). Two different magnifications are presented in the left (20×; scale bar: 100 µm) and right column (40×; scale bar: 50 µm).</p

Results^$ obtained from the screening experiments for seeding densities.

<p><i>CP: cultivation period;</i></p><p><i>SD: seeding density.</i></p>$<p><i>For a better overview, only results from one preparation were shown for 5 days of cultivation.</i></p>§<p><i>spheroidal morphology with no or short neurites (restricted neuronal morphology);</i></p><p><i><u>Confluency:</u> rated confluency in per cent; optical evaluation of the whole well under bright-field conditions using a 40-fold magnification;</i></p><p><i><u>Cell debris, conglomerates, and neuronal yield</u>.</i></p><p><i>−: no debris, conglomerates, or neurons;</i></p><p><i>+: poor amount; +(+): poor-middle;</i></p><p><i>++: middle amount; ++(+): middle-high;</i></p><p><i>+++: high amount.</i></p

Representative AFM images of individual, dissociated IC cells.

<p>AFM error signal (1) and height (2) images of four morphologically different cells (A–D) are presented. For possible cell type specification, the reader is referred to Chapter 3.2, AFM results. Arrowhead and asterisk denote the nucleus. Scale bar: 20 µm.</p

Synoptic representation of dissociation kits.

<p><i>NTDK = Neural Tissue Dissociation Kit (from Miltenyi Biotech);</i></p><p><i>Each assay includes at least two independent preparations with 2–10 wells;</i></p>$<p><i> = supplemented Panserin 401;</i></p>*<p><i> = supplemented MACS® Neuro Medium.</i></p

Anatomical overview for extraction of IC.

<p>With forceps fixed head of a neonatal rat (P3). After flapping the skin rostrally (top) and removing the calvaria, the following overview is given (from rostral to caudal): forebrain, midbrain, and hindbrain. The midbrain consists of the corpora quadrigemina, which are composed of the two rostral superior colliculi and the two caudal inferior colliculi (IC). The IC were elevated utilizing a Dumont No. 5 forceps (left).</p

Results obtained with the proteolytic enzymes.

<p><i>No formation of cell clusters was observed.</i></p><p><i><u>Confluency:</u> rated confluency in per cent; optical evaluation of the whole well under bright-field conditions using a 40-fold magnification;</i></p><p><i><u>Cell debris, conglomerates, and neuronal yield</u>.</i></p><p><i>−: no debris, conglomerates, or neurons;</i></p><p><i>+: poor amount; +(+): poor-middle;</i></p><p><i>++: middle amount; ++(+): middle-high;</i></p><p><i>+++: high amount.</i></p

Fluorescent images from the testing of proteolytic enzymes.

<p>Cells digested either with trypsin (15 min: first row) or papain (30 min: second row and 90 min: third row) were triturated without (left column) and with DNase I (right column). Merged pictures of cells labelled with TUJ1 antibody (green) and GFAP antibody (red) were depicted. When comparing all depicted conditions, an improved cytoarchitecture with high neuronal (and glial) yield was obtained in cultures dissociated with papain for 30 min without DNase I. Scale bar: 100 µm.</p

Immunocytochemical results of different dissociation protocols and media.

<p>Improved neuronal yield and branching was observed in cells cultivated for 5 days with MACS® Neuro Medium (right column). By contrast, cells cultivated with Panserin 401 (left column) showed less branching with prominent neurite fragmentation (A, E). IC tissue was dissociated with different protocols: NTDK T (A, B), NTDK P (C, D) and NTDK PN (E, F) as well as SGN-protocol (G, H). After fixation and labelling with TUJ1 (green), poor neuronal yield was obtained after dissociation with NTDK T independent from the cultivation medium. Scale bar: 100 µm.</p