In vitro characterization of human dental pulp stem cells isolated by three different methods

OBJECTIVES: In this study, we characterized human dental pulp cells (HDPCs) obtained by different culture methods to establish the most suitable methodology for dental tissue engineering and regenerative endodontic applications. MATERIALS AND METHODS: HDPCs were isolated by the outgrowth method (HDPCs-OG), the enzymatic digestion method (collagenase/dispase/trypsin, HDPCs-ED), or the combination of both methods (HDPCs-Combined). The expression of mesenchymal stem cell markers (CD105, CD90, and CD73) was investigated. In vitro differentiation capacities of HDPCs into adipogenic, osteogenic, and chondrogenic lineages were compared. Differentiation markers were analyzed by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and western blotting. RESULTS: Our data indicated that whole HDPCs-ED, HPDCs-OG, and HDPCs-Combined could be differentiated into adipogenic, chrondrogenic, and osteogenic cell types. However, we found that the methods for isolating and culturing HDPCs influence the differentiation capacities of cells. HDPCs-OG and HDPCs-ED were preferably differentiated into adipogenic and osteogenic cells, respectively. Differentiation markers shown by RT-PCR and western blotting analysis were mostly upregulated in the treated groups compared with the control groups. CONCLUSIONS: Our findings confirmed that cell populations formed by two different culture methods and the combined culture method exhibited different properties. The results of this study could provide an insight into regenerative endodontic treatment using HDPCs.

Effect of the Isolation Method of Mouse Inner Cell Mass, Types of Feeder Cells and Treatment Time of Mitomycin C on the Formation Rate of ICM Colony / 대한불임학회지

OBJECTIVE: This study was carried out to evaluate the effect of the isolation methods of inner cell mass from mouse blastocyst, types of feeder cells and treatment time of mitomycin C on the formation rate of ICM colony. METHODS: The inner cells were isolated by conventional immunosurgery, partial trophoblast dissection with syringe needles and whole blastocyst co-culture method. Commercially available STO and primary cultured mouse embryonic fibroblast (pMEF) feeder cells were used, and mitomycin C was treated for 1, 2 or 3 hours, respectively. The formation rate of ICM colony was observed after isolation of ICM and culture of ICM on the feeder cells for 7 days. RESULT: The ICM colony formation rate on STO were significantly higher in partial trophoblast dissection group (58%) than that in immunosurgery (12%) or whole blastocyst culture (16%) group (p<0.05). The formation rate on pMEF feeder layer was higher in partial trophoblast dissection (88%) and whole blastocyst culture (82%) group than that in immunosurgery (16%) group (p<0.05). When mitomycin C treated to pMEF for 2 hours, the formation rate of 88% was significantly higher than those of other conditions. CONCLUSIONS: Above results showed that the efficient isolation method of ICM from blastocyst was the partial trophoblast dissection and the appropriate treatment time of mitomycin C was 2 hours. However, the subculture of ICM colony and characterization of stem cells should be carried out to confirm the efficacy of the partial trophoblast dissection method.

Comparative study for Isolation of Yersinia pseudotuberculosis from Water / 대한임상미생물학회지

BACKGROUND: Yersinia pseudotuberculosis is recognized throughout the world as a cause of water-or food born infections in human and animals. Although many attempts have been made to define optimal conditions for the isolation of the organism from water, their isolation yields remain low; therefore, we tried to find an effective method for the recovery of Y. pseudotuberculosis from water. METHODS: Water samples were deliberately contaminated with Y. pseudotuberculosis at various levels and then processed by the following three isolation METHODS: centrifugation, direct filtration, and intracellular culture. For the centrifugation method, the water samples were centrifuged at 5000 rpm for 1 hr and the final precipitates were inoculated in cefsulodin-irgasan-novobiocin(CIN) media. For the filtration method, the water samples were filtered by negative pressure and the filter papers were put directly on CIN media. For the intracellular culture method, the organisms were extracted from the HeLa cells that had been infected with Y. pseudotuberculosis and inoculated on CIN media. We also examined the efficacy of the filtration method after cold enrichment with a mixture of Y. pseudotuberculosis, Escherichia coli, and Citrobacter freundii. RESULTS: With the concentration of 3x10(2)/100 mL, Y. pseudotuberculosis was isolated only by the filtration method; however, none of the culture methods were good enough to recover the organism from the water sample when the concentration was 3x10/100 mL. With cold enrichment, however, the recovery was much more efficient; the organism grew after direct inoculation or after filter inoculation when the starting concentrations were 3x10(2)/100 mL or 3x10/100 mL, respectively. CONCLUSION: A combined use of direct filtration and filter inoculation after cold enrichment is the most effective method to yield Y. pseudotuberculosis isolation. The introduction of effective methods for the isolation of Y. pseudotuberculosis from untreated drinking water would increase the awareness by the public of the health hazard of spring water.

Comparison with Several Methods to Isolate Epiphytic Bacteria from Gracilaria lemaneiformis (Rhodophyta) / 微生物学通报

We used 4 methods,such as ultrasonic crush(UC),ultrasonic rinse(UR),whorl surge(WS)and rubbing(RU),to isolate epiphytic bacteria from red alga Gracilaria lemaneiformis.Then,we counted bacteria numbers,detected bacterial species,observed bacterial configuration and characteristic of cell wall.Compared with these methods and with different treatments in one method,the results were drawn:the UR and RU were inferior in all methods to isolate bacterial numbers and species,the UC and WS were better,especially,the treatment 30W 30s of UC was the best in experiment,which isolated 12 of 16 bacterial species,and got 1.75 10~6 cells per gram wet weight G.lemaneiformis.

A New Method of Armillaria mellea Isolation-Gastrodia elata Tissue Isolating Method / 微生物学通报

This paper reported a new method of Armillaria mellea isolation-Gastrodia elata tissue isolating. Compared with normal isolating method-rhizomorph isolating method, it showed that the success rate of new method (78% ) was higher than the rhizomorph isolating method (16% ) , besides this, the new method was easier, and growth characteristic of obtained strain was superior to that obtained from rhizomorph isolating method.