Embryoid Body-Explant Outgrowth Cultivation from Induced Pluripotent Stem Cells in an Automated Closed Platform.

Automation of cell culture would facilitate stable cell expansion with consistent quality. In the present study, feasibility of an automated closed-cell culture system "P 4C S" for an embryoid body- (EB-) explant outgrowth culture was investigated as a model case for explant culture. After placing the induced pluripotent stem cell- (iPSC-) derived EBs into the system, the EBs successfully adhered to the culture surface and the cell outgrowth was clearly observed surrounding the adherent EBs. After confirming the outgrowth, we carried out subculture manipulation, in which the detached cells were simply dispersed by shaking the culture flask, leading to uniform cell distribution. This enabled continuous stable cell expansion, resulting in a cell yield of 3.1 × 10(7). There was no evidence of bacterial contamination throughout the cell culture experiments. We herewith developed the automated cultivation platform for EB-explant outgrowth cells.

Method for selective quantification of adipose-derived stromal/stem cells in tissue.

Fat grafts are valuable for soft-tissue regeneration and augmentation. However, fat graft systems require further improvement for the prediction of graft retention. The concentration of adipose-derived stromal/stem cells (ASCs) is one of the most important factors that affect graft retention; however, current cell quantification techniques have not been applied to adipose tissue. Here we developed a method for the selective quantification of ASCs in tissue (SQAT). We identified a characteristic methylated site in the CD31 promoter after searching for specific markers of ASCs. This DNA methylation was not detected in any cell type other than ASCs in adipose tissue. Therefore, analyzing this methylation may be a suitable approach for quantifying ASCs in tissues because DNA is readily extracted from tissues. SQAT is based on quantifying this methylation by quantitative polymerase chain reaction using methylation-sensitive HapII-treated DNA as the template. SQAT was validated based on the numbers of ASCs determined by CD31-/CD34+-based flow cytometry. The results obtained by both methods were perfectly correlated, thereby demonstrating that SQAT is a useful tool for quantifying ASCs. SQAT analysis using ASCs isolated from suctioned fat according to the standard protocol (i.e., collagenase treatment) showed that the yield of ASCs was 59% ± 21%, which suggests that the ASC isolation technique requires further improvement. Furthermore, SQAT is an excellent method for quantifying ASCs in arbitrary samples (particularly tissue), which could dramatically improve ASC isolation technologies and fat graft systems, thereby facilitating the prediction of graft retention.

Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering.

The development of an automated cell culture system would allow stable and economical cell processing for wider clinical applications in the field of regenerative medicine. However, it is crucial to determine whether the cells obtained by automated culture are comparable to those generated by manual culture. In the present study, we focused on the primary culture process of bone marrow stromal cells (BMSCs) for bone tissue engineering and investigated the feasibility of its automation using a commercially available automated cell culture system in a clinical setting. A comparison of the harvested BMSCs from manual and automated cultures using clinically acceptable protocols showed no differences in cell yields, viabilities, surface marker expression profiles, and in vivo osteogenic abilities. Cells cultured with this system also did not show malignant transformation and the automated process was revealed to be safe in terms of microbial contamination. Taken together, the automated procedure described in this report provides an approach to clinical bone tissue engineering.

In vitro evaluation of dextran sulfate cellulose beads for whole blood infusion low-density lipoprotein-hemoperfusion.

We describe results from a feasibility study of a newly developed low-density lipoprotein (LDL) adsorbent designed for use in whole-blood infusion LDL-hemoperfusion. The adsorbent has almost the same chemical structure as the Liposorber adsorbent (dextran sulfate cellulose beads) but has a larger particle size. In whole-blood perfusion tests, the adsorbent adsorbed atherogenic LDL cholesterol directly from whole blood but left concentrations of high-density lipoprotein cholesterol largely unchanged. In whole-blood perfusion tests using fresh human donor blood or bovine blood anticoagulated with acid citrate dextrose solution or sodium citrate, the adsorbent showed minimal side effects in terms of blood cell activation, complement activation, and blood cell loss, suggesting that it has excellent blood compatibility. In addition, the adsorbent showed mechanical stability and absence of hemolysis. In conclusion, the new adsorbent showed the appropriate characteristics for an LDL adsorbent column for use in whole-blood infusion LDL-hemoperfusion.

Substrate recognition and fidelity of strand joining by an archaeal DNA ligase.

We have previously identified a DNA ligase (LigTk) from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. The enzyme is the only characterized ATP-dependent DNA ligase from a hyperthermophile, and allows the analysis of enzymatic DNA ligation reactions at temperatures above the melting point of the substrates. Here we have focused on the interactions of LigTk with various DNA substrates, and its specificities toward metal cations. LigTk could utilize Mg2+, Mn2+, Sr2+ and Ca2+ as a metal cation, but not Co2+, Zn2+, Ni2+, or Cu2+. The enzyme displayed typical Michaelis-Menten steady-state kinetics with an apparent Km of 1.4 microm for nicked DNA. The kcat value of the enzyme was 0.11*s-1. Using various 3' hydroxyl group donors (L-DNA) and 5' phosphate group donors (R-DNA), we could detect ligation products as short as 16 nucleotides, the products of 7 + 9 nucleotide or 8 + 8 nucleotide combinations at 40 degrees C. An elevation in temperature led to a decrease in reaction efficiency when short oligonucleotides were used, suggesting that the formation of a nicked, double-stranded DNA substrate preceded enzyme-substrate recognition. LigTk was not inhibited by the addition of excess duplex DNA, implying that the enzyme did not bind strongly to the double-stranded ligation product after nick-sealing. In terms of reaction fidelity, LigTk was found to ligate various substrates with mismatched base-pairing at the 5' end of the nick, but did not show activity towards the 3' mismatched substrates. LigTk could not seal substrates with a 1-nucleotide or 2-nucleotide gap. Small amounts of ligation products were detected with DNA substrates containing a single nucleotide insertion, relatively more with the 5' insertions. The results revealed the importance of proper base-pairing at the 3' hydroxyl side of the nick for the ligation reaction by LigTk.