Article Figures & Data
- Figure 1.
Experimental design and work-flow. Submucous plexus viability was analyzed in six groups of tissues, which were stored in different cryopreserving conditions. Structural and neuron numbers were assessed through H&E and immunostainings, and neuronal viability with Ca2+ imaging. The chart also indicates the number of biopsies (n) subjected to each experimental procedure. CP, cryopreservation; FI, fresh isolation; DMSO, Dimethyl sulfoxide; D, day; W, week; M, month; Y, year; NBM, Neurobasal medium.
- Figure 2.
Scheme of protocol for sample processing. Biopsies were collected and placed at 4°C for no more than 30 min before starting the dissection. Once the submucous plexus is dissected, it is placed in NBM with 8% DMSO in isopentane and transferred to a −80°C freezer for 5–10 h. Biopsies are then, for longer storage, placed in liquid nitrogen for up to one year. Thawing was quickly performed, once ice is melted biopsy is changed into fresh NBM at 37°C. NBM, Neurobasal medium.
- Figure 3.
Representative images of H&E, NSE (black), and S100 (white) immunohistochemical stainings colonic biopsies, before cryopreservation conserved in NBM and after one-week cryopreservation with and without DMSO. H&E, NSE (brown), and S100 (red) images are shown as labeled on the left. The tissue sections (epithelium, lamina propria, or submucosa) are labeled on the right. The first column shows freshly isolated and fixed biopsies, second column shows biopsies fixed after one week of cryopreservation with NBM, third column shows biopsies fixed after one week of cryopreservation with NBM and DMSO. For each staining, the black dashed regions are shown magnified in the row below. Scale bars: 20 μm. White arrows point at S100-positive glial cells, and black arrows show NSE-positive neurons.
- Figure 4.
Temperature curves for freezing and thawing procedures. All conditions with isopentane full lines; without isopentane, dashed lines. MilliQ water in blue; NBM without DMSO in green; NBM with DMSO in orange. Note that isopentane slows the freezing process over 5-fold independent of the medium (gray double headed-arrow); furthermore, the addition of DMSO shifts freezing curves moderately to the left, specifically in the −5°C range (shaded bar). The effects of either DMSO or isopentane during thawing are less pronounced. DMSO, Dimethyl sulfoxide; NBM, Neurobasal medium; w, with; w/o, without.
- Figure 5.
A, Representative images of immunohistochemical stainings of dissected submucous ganglia. Submucosal neurons (HuCD) in green and nerve fibers (NF200) in red. Immunostainings were performed either immediately (a) or after cryopreservation for different time periods: 1 d without (D1w/oD; b) or with (D1wD; c) DMSO, one week (W1wD; d), three months (M3wD; e) or one year (Y1wD; f). Magnification of the selected ganglion (square) from top panel is in the bottom panel. Scale bar: 50 μm. B, C, Quantification of submucosal neurons. B, Average number of neurons per ganglion. C, Percentage of neurons with nuclear HuCD. The individual data points represent a subject for which all individual neuronal responses were averaged. Horizontal bars represent the median. Comparison between groups was performed using Kruskal–Wallis test, *p < 0.05, **p < 0.01.
- Figure 6.
Ca2+ imaging in human biopsies. A, FI SMP recording and matched post hoc immunostaining. A1, Representative image of a SMP ganglion loaded with Fluo-4. Color-coded arrows point at two neurons, whose Ca2+ traces are shown in A2. Scale bar: 50 μm. A2, Normalized fluorescence profiles (ΔF/F0) of the two neurons marked in A. A3, The submucous ganglion (shown in A1) fixed and immunostained for HuCD (green) and NF200 (red). A4, Larger magnification of the dashed box. Scale bar: 50 μm. B, Representative image of a SMP ganglion after three months of cryopreservation loaded with Fluo-4. ROIs mark three different neurons whose Ca2+ traces are shown on the right. The actual recording corresponds to Movie 1. Scale bar: 50 μm. C, Representative image of a SMP ganglion after one year of cryopreservation loaded with Fluo-4. ROIs mark three different neurons whose Ca2+ traces are shown on the right. The actual recording corresponds to Movie 2. Scale bar: 50 μm. D, Summary data of the neuronal responses to three different stimuli for six different conditions. Top row shows the percentage of responding neurons and bottom row the peak amplitude after stimulation with high-K+, 5-HT (serotonin) and DMPP (dimethylphenylpiperazinium). The individual data points represent a subject for which all individual neuronal responses were averaged. Horizontal bars represent the median. Comparison between groups was performed using Kruskal–Wallis test, *p < 0.05, **p < 0.01.
Abbreviation Cryopreservation NBM complemented
with DMSOFI Freshly isolated tissue No D1w/oD 1 day cryopreserved No W1w/oD 1 week cryopreserved No D1wD 1 day cryopreserved With 8% DMSO W1wD 1 week cryopreserved With 8% DMSO M3wD 3 months cryopreserved With 8% DMSO Y1wD 1 year cryopreserved With 8% DMSO NBM, Neurobasal medium; DMSO, Dimethyl sulfoxide
n No damage (grade 1–2) Moderate damage (grade 3–4) Severe damage (grade 5–6) Fresh isolation 6 100% 0% 0% W1w/oD 3 100% 0% 0% W1wD 3 100% 0% 0% Values are expressed as percentage (%)
Condition Preservation medium Cooling/freezing W/WO isopentane Storage Responding cells (%) 1 NBM 4°C - 4°C > 6 h 0% 2 NBM −20°C WO −20°C 0% 3 NBM −80°C WO −80°C 0% 4 NBM + 8% DMSO Snap Liq N2 WO Liq N2 0% 5 NBM + 8% DMSO −20°C, 10 min WO Liq N2 0% 6 NBM + 8% DMSO −80°C, 24 h WO Liq N2 0% 7 NBM −80°C, 5–10 h W Liq N2 <50% 8 NBM + 8% DMSO −80°C, 5–10 h W Liq N2 >50% NBM, Neurobasal medium; DMSO, Dimethyl sulfoxide; Liq N2, Liquid nitrogen; W, with; WO, without.
- Movie 1.
Representative recording of high-K+ Krebs stimulation of the colonic ganglion Fluo-4 loaded after one year of cryopreservation shown in Figure 6. A 1-min recording at 2 Hz. Timescale shown at top left. Upon high-K+ Krebs stimulation (between second 20 and 30, as shown by the hK mark below the timescale), a ganglion response can be observed. Scale bars: 20 μm.
- Movie 2.
Representative recording of high-K+ Krebs stimulation of the colonic ganglion Fluo-4 loaded after three months of cryopreservation shown in Figure 6. A 1-min recording at 2 Hz. Timescale shown at top left. Upon high-K+ Krebs stimulation (between second 20 and 30, as shown by the hK mark below the timescale), a ganglion response can be observed. Scale bars: 20 μm.
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