Cell viability after cold storage of primary rat hepatocytes in different solutions.

<p>(A) Progression of cell death during cold storage (4°C) in cell culture medium and Krebs-Henseleit-buffer (KH). (B) Cell viability after one week of cold storage in cell culture medium, cold storage solutions 1 and 2 (n = 7;* p = 0.0003). (C-F) Original FACS plots showing cell viability after one week of cold storage. Cells were stained with propidium iodide (PI, 5 µg/mL) and viability was assessed by flow cytometry. C: Non-stored control cells. Cells after cold storage in cell culture medium (D), chloride-rich solution 1 (E) or chloride-poor solution 2 (F). Red dots indicate dead (i.e. propidium iodide-positive) cells. The percentage of viable cells is given in each panel.</p

Cell attachment after one week of cold storage in different solutions.

<p>(A) Control culture of non-stored primary rat hepatocytes. (B-D) Cell cultures from cell suspensions stored for one week in cell culture medium (B), chloride-rich, potassium-rich solution 1 (C) and chloride-poor solution 2 with balanced Na⁺/K⁺ concentrations (D). (E) Quantification of adherent, viable cells after cold storage and 24 h of cell culture as percentage of cultures of non-stored control cells. (F) Reductive metabolism of cultures obtained from cold-stored cell suspensions, given as percentage of non-stored control cell cultures (n = 8, *p<0.0001).</p

Comparison of solutions 2 and 6 to established organ preservation solutions.

<p>Viability (A), cell attachment (B) and resazurin conversion (as measure for general reductive metabolism, C) after one week of cold storage in solution 2, University of Wisconsin solution (UW), histidine-tryptophan-ketoglutarate solution (HTK) and Celsior (n = 8). Urea production (D) and forskolin-triggered glucose release (E) of cultures from cells stored in solutions 2 and 6, UW and Celsior for one week (n = 6). Activities are expressed as percentage of cultures from non-stored control cells; *p≤0.0001.</p

Influence of ion composition and iron chelators on cell volume.

<p>Histograms of cell volume distribution as assessed by flow cytometry after one week of cold storage in different solutions. Storage in cell culture medium induced marked cell swelling (A), storage in chloride-rich solutions containing iron chelators slight swelling (B, C). Cell swelling in chloride-rich basic solutions was as pronounced as in cell culture medium, irrespective of sodium concentration (D), substitution of chloride by lactobionate induced marked cell shrinkage (B, C). Cell swelling was inhibited by the addition of iron chelators (E, F; basic solution 3 vs. solution 3 w/o adenosine, solution 3 vs. basic solution 3+ adenosine), but not influenced by addition of adenosine (E, F).</p

Influence of cold storage solution components on cell viability after cold storage.

<p>‘Basic solution’ refers to solutions without iron chelators (deferoxamine + LK 614) and without adenosine. In supplemented basic solution 7, NaCl addition was reduced to maintain osmolarity. Data is given as median (25/75% percentile).</p>*<p>significantly different from cell culture medium.</p>†<p>significantly different from basic solution 7 (n = 6).</p

Composition of cold storage solutions.

<p>KH: Krebs-Henseleit buffer; L-15: Leibovitz L-15 cell culture medium.</p><p>All concentrations are given in mM. Cold storage solutions 1–7 without iron chelators (LK 614 and deferoxamine) and without adenosine are referred to as <b>‘basic solutions’</b> in the text. L-15 cell culture medium contains amino acids, trace elements etc. not listed in this table (to maintain lucidity) and was additionally supplemented (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040444#s2" target="_blank">Materials and Methods</a>) prior to use. Calculated osmolarity is given in mosm/L.</p>*<p>Before use, calculated osmolarity was raised to 311 mosm/L (basic solution: 305.2 msom/L) by addition of NaCl.</p

Four CEACAM1 splice variants are expressed in confluent A549 cells.

<p>RT of RNA isolated from confluent A549 cells were applied to four different PCR reactions using primer pairs specific four each of the four CEACAM1 isoforms. Products corresponding to CEACAM1-4L (266 bp), CEACAM1-4S (245 bp), CEACAM1-3L (177 bp), and CEACAM1-3S (145 bp), respectively, were amplified. The size of oligonucleotide markers is shown on the left.</p

Primer sequences used in RT-PCR.

<p>Primer sequences used in RT-PCR.</p

CEACAM6 acts as an inducer of cellular proliferation in confluent A549.

<p>A) The expression of the proliferation marker Ki67 is limited to A549-T cells that also expressed CEACAM6 on their cell surface. Confluent CEACAM6-negative and CEACAM6-positive A549-T cells were separated by mAb 9A6 loaded magnetic protein G microbeads and μMAS magnetic sorting columns (Miltenyi Biotec) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008747#s4" target="_blank">Materials and Methods</a>. Immunoblot analysis of confluent CEACAM6-negative and CEACAM6-positive A549-T cell lysates was performed applying a Ki67 specific antibody followed by HRP-coupled secondary antibody and ECL detection. Beta-actin served as a loading control. The data shown are representative of three separate experiments. B) Cell cycle analysis of CEACAM6 negative and CEACAM6 positive A549-T cells. CEACAM6-negative and CEACAM6-positive A549 cells were fixed in 80% ethanol and stained with propidium iodide, and analyzed by flow cytometry as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008747#s4" target="_blank">Materials and Methods</a>.” The DNA content in the different cell fractions is given in arbitrary units on the X-axis. Cells in the G2-M phase (second peak) contained twice as much DNA as cells in the G0-G1 phase (first peak). Cells between the peaks represent cells in the S-phase. The relative proportions of cells in the various phases are shown above the DNA profiles. Filled curve, CEACAM6 negative A549 cells; thick curve, CEACAM6 positive A549 cells. C) Confluent control sh-plasmid transfected A549-T (A549-shControl) and shCEACAM6 transfected A549-T cells (A549-shCC6) were stained for CEACAM1 with mAb clone 283340 and for CEACAM6 with mAb 9A6 (thick lines). The background fluorescence was determined by incubating the cells with control IgG antibody (thin lines). Samples were analyzed by flow cytometry. Compared to A549-shControl cells, A549-shCC6 cells completely lacked CEACAM6 expression, but continued to express CEACAM1. D) Phase contrast images of (a) control sh-plasmid transfected A549-T and (b) shCEACAM6 transfected A549-T cells. Confluent control sh-plasmid transfected A549-T piled up and formed unanchored spheroidal cell aggregates on top of the monolayer revealing insufficient contact inhibition. (b) In contrast, A549-shCC6 cells formed well spread monolayers without detection of unanchored, spheroidal cell growth indicative tight contact inhibition. Bar, 50 µm.</p

CEACAM1 expression differs in contact-inhibited and proliferating A549 cells.

<p>A) For quantification of CEACAM1 cell surface expression in confluent and log phase cultured A549 cells, samples were analyzed by flow cytometry utilizing the QuiFiKit approach as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008747#s4" target="_blank">Materials and Methods</a>. Briefly, cells were stained for CEACAM1 with mAb clone 283340. Fluorescence was quantitated using QuiFiKit calibration-beads as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008747#s4" target="_blank">Materials and Methods</a>. The data shown are means ± SD (*p≤0.007) of three different experiments. B) Immunoblot analyses of confluent and proliferating A549 cell lysates were done as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008747#s4" target="_blank">Materials and Methods</a> using monospecific mAbs directed against CEACAM1 (clone 283340), CEACAM5 (Col-1), and CEACAM6 (9A6), respectively and visualized by HRP-coupled secondary antibody and ECL detection. Beta-actin served as a loading control. The blots shown are representative of three separate experiments. C) The fraction of CEACAM1 expressing A549 cells increased over time as cells were kept in the confluent state. To analyze the CEACAM1 expression in A549 cells grown to confluence (day 0), plus 1 day, plus 3 days, plus 5 days and plus 7 days, cells were stained with mAb clone 283340 (thick line) and isotype matched control antibody (thin line) followed by FITC-conjugated secondary antibody. Subsequently, samples were measured by flow cytometry. The data shown are representative of three different experiments.</p