Microchannel formation by spatially bioprinting the sacrificial living porogens using a cell bioprinter.

<p>(<b>a</b>) <i>E. coli</i> (800,000 CFU/mL) were mixed with 0.5% agarose at 40°C. Agarose-bacteria mixture was printed on a 1% agarose pre-coated petri dish and covered with another 1% agarose layer on top. (<b>b</b>) Top view of bacterial colony chain in 0.5% agarose. (<b>c</b>) Merged bacterial colonies. (<b>d</b>) Cross-section of a formed microchannel in the hydrogel. (<b>e</b>) Diffusion enhanced in bioprinted microfluidic hydrogels at areas with high porous density.</p

Illustration of the fabrication steps of microporous hydrogel scaffolds using living sacrificial porogens.

<p>(<b>a</b>) <i>E. coli</i> encapsulation in hydrogels as porogens and l<b>iv</b>e sacrificial pore formation. <i>E. coli</i> cultured on LB agar plate were collected and mixed with the agarose solution. After mixing, <i>E. coli</i> suspension was poured into a 12-well plate and solidifies. <i>E. coli</i> encapsulated in hydrogels were continuously cultured to allow formation of colonies. The living porogens were then lysed and the debris of <i>E. coli</i> and its DNA were removed by sequential washing with DPBS and DI water. (<b>b</b>) Formation of microfluidic channels. A line of <i>E. coli</i> / agarose mixture solution was printed onto Petri dish pre-coated with a layer of agarose. Then, another layer of agarose was used to cover the bacterial line. The hydrogels were gelled under rapid cooling (4°C) overnight.</p

The mechanical stiffness (a–b) and perfusion properties (c–f) of porous hydrogels.

<p>Compressive moduli were inversely correlated with the culture time for 1% hydrogel (<b>a</b>), whereas there was no significant change in stiffness for 2% hydrogel (<b>b</b>). Fluorescence images of FITC-dextran (0.25 mM, 20 kDa) diffusion in the 1% porous agarose hydrogels (<b>c</b>) and controls (<b>d</b>). The diffusion profiles of FITC-dextran as a function of distance from the source channel in porous (<b>e</b>) and non-porous (<b>f</b>) hydrogels. (n = 3).</p

Characterization of living porogen growth in hydrogels and subsequent pore formation.

<p>Crystal violet staining of bacterial colonies in 1% (<b>a</b>) and 2% (<b>b</b>) hydrogels. SEM images of pores created using living porogens (<b>c</b>) as compared to controls (<b>d</b>). The colony size (<b>e–f</b>), density (<b>g–h</b>), and the pore area percentage (<b>i–j</b>) are presented over the culture time for 1% and 2% hydrogels at initial bacterial seeding concentrations of 9.5×10⁷, 1.9×10⁸ and 3.8×10⁸ CFUs/ml.</p

Biocompatibility of fabricated porous hydrogels.

<p>After lysis of porogens, the porous scaffolds were washed with DPBS and cell medium. 3T3 were then seeded on the scaffold and cultured. 3T3 cells proliferated and were confluent on day 6 for 1% hydrogel (<b>a–c</b>) and on day 14 for 2% hydrogel (<b>d–f</b>). Cells were alive after confluence (<b>g–h</b>).</p

Multiplex-PCR primers designed for the amplification of <i>H</i>. <i>pylori</i> virulence genes.

<p>Multiplex-PCR primers designed for the amplification of <i>H</i>. <i>pylori</i> virulence genes.</p

Expert-derived models for diagnostic prediction of gastric diseases using <i>H</i>. <i>pylori</i> virulence factors and host immune responses.

<p><b>(a)</b> shows that a possible relationship prediction between the <i>vacAm1/m2</i>, <i>cagA</i> and <i>ureA</i> and IL-17, FOXP3 and patient’s clinical outcomes and <b>(b)</b> shows that the possible relationship prediction between <i>vacAm1/m2</i>, <i>cagA</i> and <i>ureA</i> and IL-17, FOXP3 and IFN-γ and patient’s clinical outcomes.</p

Quantitative RT-PCR primers designed for the detection of Th1, Th17 and Treg cell response.

<p>Quantitative RT-PCR primers designed for the detection of Th1, Th17 and Treg cell response.</p

Multiplex-PCR Assay For <i>Helicobacter</i>-specific Virulence Factors.

<p><b>(a)</b> amplification of virulence genes of <i>H</i>. <i>pylori</i> G27 strain by multiplex PCR; Lane M, 100 bp- marker (ThermoSCIENTIFIC, Gene Ruler), Lane 1 (Reaction 1); <i>ureA</i> (244bp), <i>ureB</i> (645bp), <i>hpaA</i> (534bp), <i>cagA</i> (415bp), <i>napA</i> (384bp), Lane 2 (Reaction 2); <i>dupA</i> (584bp), <i>oipA</i> (401bp), <i>vacA</i> (333bp), and Lane 3 (Reaction 3); <i>babA</i> (832bp) <b>(b)</b> PCR inferred results of <i>s1/s2</i> and <i>m1/m2</i> allelles of <i>vacA</i> gene; Lane M, 100 bp- marker (ThermoSCIENTIFIC, Gene Ruler), Lane 1 (reaction 1); <i>vacA s1</i> (259bp), Lane 2 (Reaction 2); <i>vacA s2</i> (286 bp), Lane 3 (Reaction 3); <i>vacA m1</i> (567 bp), and Lane 4 (Reaction 4) <i>vacA m2</i>(642 bp). <b>(c)</b> Multiplex-PCR application of the biopsy samples taken from randomly selected patients with ulcer and gastritis; Lane M, 100 bp- marker (ThermoSCIENTIFIC, Gene Ruler), between Lane 1 to 6 are representing patients with gastritis or ulcer: Multiplex PCR reaction results of patient 1 and 2 (gastritis or ulcer) are shown between Lane 1 to 3 and Lane 4 to 6, respectively. Reaction 1 (Lane 1 and 4) <i>ureA</i> (244bp), <i>ureB</i> (645bp), <i>hpaA</i> (534bp), <i>cagA</i> (415bp), <i>napA</i> (384bp), Reaction 2 (Lane 2 and 5); <i>dupA</i> (584bp), <i>oipA</i> (401bp), <i>vacA</i> (333bp), and Reaction 3 (Lane 3 and 6); <i>babA</i>(832bp) <b>(d)</b> PCR inferred results of <i>s1/s2</i> and <i>m1/m2</i> allelles of <i>vacA</i> gene of randomly selected patients with gastritis and ulcer respectively; Lane M, 100 bp- marker (ThermoSCIENTIFIC, Gene Ruler), between Lane 1 to 3 are representing patients with gastritis (left side) or ulcer (right side): Patients with gastritis; Lane 1–3, <i>vacA s1</i> (259bp), and <i>vacA s2</i> (286 bp), patients with ulcer; Lane 1–3, <i>vacA m1</i> (567 bp), and <i>vacA m2</i> (642 bp). <b>(e)</b> Multiplex urease-PCR assay to detect the <i>Helicobacter</i> positive and negative samples; Lane M, 100 bp- marker (ThermoSCIENTIFIC, Gene Ruler), Lane 1–2 are from randomly selected patients, and lane 3 is from <i>H</i>.<i>pylori</i> positive control strain G27. Sybr Gold (Invitrogene) was used for the gel in (a) and (c) and gel pictures were taken by using Observable Real Time Gel Electrophoresis System (Salubris Technica, Turkey).</p