JNK3 expression analysis.

<p><i>(</i><b><i>a/b</i></b><i>)</i> Protein extracts (30 µg) were prepared from tissue of different species (human, pig and mouse): skeletal muscle (S. Muscle), pancreas, brain and islets generated from wild type (<i>WT</i>) and <i>Jnk3</i>-knockout (<i>KO</i>) mice. JNK3 expression levels were examined by Western blot in <i>D₀</i> and <i>D₇</i> using a JNK3 monoclonal antibody. JNK1 and tubulin monoclonal antibodies were used as loading controls. Protein bands were scanned and quantified by Odyssey scan software. The values were summarized in graphics. Data are means ± SEM of eighteen independent experiments (n = 18, <i>^***P<0.001</i> for JNK3 in <i>D₀</i> (black bars) <i>vs. D₇</i> (grey bars)).</p

JNK3 and c-fos strongly correlate to OCR.

<p>Correlation analysis using <i>the Spearman's</i> correlation coefficient <i>rho</i> was determined as described in <i>“Statistical analysis”</i> by plotting the values of delta <i>(d)</i>-OCR_D7-D0 together with <i>(</i><b><i>a</i></b><i>) d-JNK3</i> (n = 18, <i>^***P<0.001</i>, 0.7853) and with <i>(</i><b><i>b</i></b><i>) d-fos</i> (n = 6, 0.8857).</p

Islet culture improves islet viability by increasing ATP content and OCR.

<p><i>(</i><b><i>a</i></b><i>)</i> ATP was measured and normalized to DNA content (nmol/mg protein/mg-DNA) as described in <i>“</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099796#s2" target="_blank"><i>Materials and Methods</i></a><i>”</i>. Graphic represents ATP/DNA values. Results are means ± SEM of five separate experiments performed in triplicate (n = 5, <i>^**P<0.01</i> for <i>D₀</i> (black bars) <i>vs. D₇</i> (grey bars)). <i>(</i><b><i>b</i></b><i>)</i> Islet samples were used for OCR measurements normalized to DNA content (nmolO₂/min/mg-DNA) as described in <i>“</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099796#s2" target="_blank"><i>Materials and Methods</i></a><i>”</i>. Graphic represents OCR/DNA values. Data are means ± SEM of eighteen separate experiments performed in triplicate (n = 18, <i>^***P<0.001</i> for <i>D₀ vs. D₇</i>). <i>(</i><b><i>c/d</i></b><i>)</i> Protein extracts (30 µg) from islet samples with <i>(</i><b><i>c</i></b><i>)</i> high or <i>(</i><b><i>d</i></b><i>)</i> low JNK3 protein content in <i>D₀</i> and <i>D₇</i> were tested by Western blot. Polyclonal antibodies against: p-Akt1, p-Akt2, p-GSK3β, JNK3, MKK7, Akts and PTEN were used. Equal protein loading was assessed by blotting membranes with antibodies against tubulin and GAPDH. Data are representative of at least three separate experiments (n = 3–8).</p

Effect of the islet isolation procedure on activation of MAPKs.

<p><i>(</i><b><i>a/b</i></b><i>)</i> For measuring JNK activation by JNK assays, protein extracts (50 µg) were mixed with the GST-Jun fusion protein (GST-c-Jun) and used as described in <i>“</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099796#s2" target="_blank"><i>Materials and Methods</i></a><i>”</i> Photographs represent ^γ33P-phosphorylation of the substrate (p-GST-c-Jun) following SDS-PAGE. The levels of <i>(</i><b><i>c/d</i></b><i>)</i> p38 and <i>(</i><b><i>e/f</i></b><i>)</i> ERK_1/2 protein phosphorylation and expression were determined by Western blot using antibodies against: phospho (p)-p38, p38, p-ERK_1/2 and ERK_1/2. The band density values were calculated as a ratio of <i>(</i><b><i>a/b</i></b><i>)</i> p-GST-c-Jun normalized to GST-c-Jun, <i>(</i><b><i>c/d</i></b><i>)</i> p-p38s normalized to p38s and <i>(</i><b><i>e/f</i></b><i>)</i> p-ERK_1/2 normalized to ERK_1/2. Results are means ± SEM of five to thirteen separate experiments (n = 5–13) and are presented as graphics: white bars; pancreas (>98% exocrine tissue), black bars; purified <i>D₀</i> islets (90% endocrine tissue) and grey bars; cultured <i>D₇</i> islets (>90% endocrine tissue). <i>(</i><b><i>a/b</i></b><i>)</i> JNK activation: Statistically significant differences are assessed by ANOVA or Student's <i>t</i>-test with <i>^*P<0.05</i> or <i>^**P<0.01</i> for all groups prior <i>vs.</i> after SW (n = 5) and for <i>D₀ vs. D₇</i> islets (n = 13). No significant differences are found among all groups from <i>T₀</i> to <i>T₁₅</i>. <i>(</i><b><i>c/d</i></b><i>)</i> p38 activation: Differences are significant for groups <i>T₀–₁₅</i> compared to <i>T_sw-₄₀</i> during pancreas digestion (n = 5, <i>^*P<0.05</i>) and for <i>D₀ vs. D₇</i> islets (n = 13, <i>^*P<0.05</i>). <i>(</i><b><i>e/f</i></b><i>)</i> No significant differences are found among all groups.</p

Design of continuous feed apparatus.

<p>(<b>A</b>) Schematic diagram of continuous feeding perfusion circuit with fresh medium, perfusion pump, outflow tube, and waste medium. Solid blue lines are medium connections, dashed green lines are fiber optic connections, and dotted black lines are electronic connections. (<b>B</b>) Exploded illustration of outflow tube showing small pores, which do not allow spheroids to pass through, but allow the removal of waste medium. The continuous feeding system was designed to help improve consistency of nutrient and supplement concentrations making β-TC6 cell culture parameters more stable and controlled.</p

Fluctuations in medium nutrient levels contributed to limitations in cell expansion.

<p>Glucose measurements from β-TC6 cell spheroids cultured in (A) Static cultures, and (B) stirred suspension bioreactors using high (4.5 g/L), intermediate (2.75 g/L), and low (1.0 g/L) glucose medium, as depicted by their position on the Y axis. The physiological glucose range is indicated by the grey bar. Error bars for glucose measurements are too small to be visible on the scale shown (Standard Error ≤4% for all measurements). (C) No difference was seen comparing static to SSB cultures with any of the glucose levels. Comparison of expansion of β-TC6 spheroid cultures indicated that changing the glucose in the medium to achieve levels closer to the physiological range did not significantly improve cell expansion. (*indicates a p value of 0.027 compared with the same culture method using high glucose medium.) SSB: stirred suspension bioreactor.</p

Stirred suspension bioreactors offered no growth improvement compared to static cultures.

<p>Fold expansion of β-TC6 spheroids after twenty one days compared stirred suspension bioreactor to static culture using standard high glucose medium.</p

Adjusting culture medium feed rate regulates glucose concentrations and improves cell growth compared to continuous feeding at a constant rate.

<p>(<b>A</b>) Cell counts comparing constant feed rate to adjusted feed rate from the same cultures (*represents a p value <0.05 indicating a significant difference in culture expansion). (<b>B</b>) Average culture medium glucose levels from 21 day constant feeding, and adjusted feeding bioreactor cultures. The physiological glucose range is indicated by the grey bar. Error bars for glucose measurements are too small to be visible on the scale shown (Standard Error ≤4% for all measurements).</p

Continuously fed SSB eliminated glucose fluctuations and improved cell expansion.

<p>(<b>A</b>) Glucose measurements for β-TC6 spheroid culture medium using static, SSB, and CF-SSB culture methods and feeding with standard high glucose medium. The physiological glucose range is indicated by the grey bar. Error bars for glucose measurements are too small to be visible on the scale shown (Standard Error ≤4% for all measurements). (<b>B</b>) Fold Expansion of β-TC6 spheroids over 21 days of culture comparing static, SSB, and CF culture methods. SSB: stirred suspension bioreactor. CF: Continuously fed stirred suspension bioreactor.</p

Continuous feeding at a constant rate cannot maintain a physiological glucose level.

<p>Culture medium glucose measurements from continuous fed cultures with high, intermediate, or low glucose medium during 21 days of expansion. The physiological glucose range is indicated by the grey bar.</p