• Journal of the korean academy of Pediatric Dentistry
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• v.44 no.3
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• pp.350-357
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• 2017

The purpose of this study was to evaluate the difference of differentiation potential in each passage of dental pulp stem cells from supernumerary tooth (sDPSCs). The sDPSCs were obtained from a healthy 6-year-old male patient under the guidelines and got the informed consent. Cells were cultured until passage number 16 and divided into two groups; 1 - 8 passages as a young group and 9 - 16 passages as an old group. It was taken $2.25{\pm}0.46days$ in a young group and $3.25{\pm}0.46days$ in an old group to propagate cells of each passage until confluence and there were statistically significant differences between two groups (p < 0.05). In every passage, cell morphology was observed with microscope and evaluated the capacity to form high levels of minerals by alizarin red solution staining after treating differentiation medium. Fibroblast-like, spindle shaped, elongated cells and a few nodules were found in uninduced cultures of passage number 1, 8 and 9. But at 16 passage culture, cell size became larger and broader and observed with more nodules. After inducing differentiation, mineralized nodules were detected at the first passage of 7th day culture whereas at the 8 passage culture, nodules were seen clearly at 14th day culture. In addition, the amount of mineralized nodules were remarkably decreased after passage 9. From the data presented in this study, it is recommended to use sDPSCs of passage number within 8 for utilizing as stem cells.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.77-77
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• 2002

본 연구는 체세포 및 체세포 핵이식란의 염색체 이상에 체세포의 계대의 영향성을 조사하고자 하였다. 한우 성축의 귀 조직으로 얻어진 세포를 공여세포를 체외성숙 후 제핵된 난자에 핵이식을 실시하였으며, 1.9kv/cm, 20$\mu\textrm{s}$/2times의 전기자극으로 융합후 5$\mu\textrm{g}$/ml의 ionomycin에서 4min, 1.9mM 6-DMAP에서 4h동안 배양함으로써 활성화를 유도하였다. 핵이식란은 CRlaa에서 4일간 배양 후 8 -cell단계에서 중기상의 유도를 위하여 상기 배양액 1ml당 0.05$\mu\textrm{g}$ colcemid에서 6-8시간 더 배양하였다. 이후, 6% Fetal bovine serum이 함유된 1% sodium citrate용액에 20분간 저장처리 후, methanol 5 : aceticacid 1 : distilled water 4로 1차, methanol 3: aceticacid 1 로 조성된 2차, methanol 4 : acetic acid 3 : distilled water 1의 3차고정액으로 1분간 재 침지시켰다. 고정 처리가 완료된 slide는 4% Giemsa용액으로 염색한 후 광학현미경 하에서 핵형 양상을 검경하였다. 체세포의 5계대에서는 684개의 spreads를 검경한 결과 염색체 수는 72%가 정상으로 60개이었고, 24%가 60개 이하였으며 4%가 60개 이상을 보였다. 10계대도 5계대와 비슷하여 71%가 정상, 26%가 60개 이하, 3%가 60개 이상이었고, 15계대에서는 55%가 정상이었고, 30%가 60개이하, 15%가 60개 이상을 보였다. 10계대 까지는 mixoploid의 비율의 변화가 없었으나 15계대에서 현저하게 늘어남을 볼 수 있었다. 또한 체외수정란과 핵이식란의 비교에서는 체외수정란은 250개의 spreads를 검경한 결과 염색체 수는 95.6%가 정상으로 60개이었고, 2.0%가 60개 이하, 2.4%가 60개 이상이었으나, 핵이식란은 204개를 검경하여 88%가 정상이었고, 4.9%가 60개이하, 7.1%가 60개 이상을 보임으로써, 핵이식란이 체외수정란에 비하여 염색체 이상의 비율이 높았다. 따라서 계대에 따라 체세포의 염색체이상의 비율이 상대적으로 증가하고, 체세포 핵이식에 따른 염색체 이상이 생길 수 있음을 알 수 있었다. (이 논문은 농림부 연구비에 의해 수행되었음)

• Journal of the korean academy of Pediatric Dentistry
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• v.45 no.4
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• pp.492-498
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• 2018

This study was conducted to investigate the characteristics of subculture times in the early, middle, and late passages by measuring the time under subculture until it was judged that the supernumerary tooth-derived pulp stem cells (sDPSCS) were no longer proliferating. Three supernumerary teeth from two healthy six-years old boys were extracted and stem cells were obtained from the pulp tissue. This was called SNT1 (supernumerary tooth 1), SNT2, and the supernumerary tooth from another child was named SNT3. SNT1 and 2 were subcultured at the same time and SNT3 was subcultured a little faster. The mean time of complete subculture was $3.6{\pm}1.1$ days. Total passages were cultured up to $23.3{\pm}0.6$ and took 83 days. These were divided into three groups based on the passage. The increase rate of time taken in subculture between group I and group II was 11.9%, but the rate between group II and group III was 28.6%, which was 2.4 times increased. The time taken between passages during long-term subculture up to 22 passages shows a regressive pattern y = 0.1169x + 2.25 and y = 0.1169x + 2.0. In conclusion, the passage time of SPSCs increased in late passages, and it shows a similar pattern.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.1
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• pp.23-34
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• 2009

Objectives: Mesenchymal stem cells (MSC) comprise a promising tool for cellular therapy. It is known that long-term in vitro culture of human bone marrow and adipose tissue derived-MSCs lead to a reduction of life span and a change of stem-like characters. The aim of our study was to examine whether stem cell properties of human umbilical cord-derived stem cells (HUC) could be affected by in vitro expansion. Methods: HUC were isolated from human umbilical cord and cultured for 10 passages in vitro. Morphology and population doubling time (PDT) were investigated, and changes of stem cell properties were examined using RT-PCR and immunocytochemistry during serial subcultures. Results: Morphology and PDT of HUC began to change slightly from the 7th passage (p7). Expression level of nestin and vimentin mRNAs increased along with the culture period from p4 until p10. In contrast, expression level of SCF mRNA decreased during the same culture period. Expression level of Oct-4 and HNF-4${\alpha}$ mRNAs was not significantly changed throughout the culture period until p10. Expression level of BMP-4, FGF-5, NCAM and HLA-ABC mRNAs appeared to increase as the culture continued, however, the difference was not significant. Immunocytochemical studies showed that HUC at p3, p6 and p9 positively were stained with antibodies against SSEA-3 and SSEA-4 proteins. Interestingly, staining intensity of HUC for ICAM-1 and HLA-ABC gradually increased throughout the culture period. Intensity against thy-1 and fibronectin antibodies increased at p9 while that against TRA-1-60 and VCAM-1 antibodies began to decrease at p6 until p9. Conclusions: These results suggest that HUC change some of their stem cell characteristics during in vitro culture. Development of culture system might be needed for the maintenance of characteristics.

• Journal of the korean academy of Pediatric Dentistry
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• v.48 no.3
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• pp.291-301
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• 2021

The aim of this study is to compare the properties of odontoblast gene of early passage cells and late passage cells derived from impacted maxillary supernumerary teeth. Impacted supernumerary teeth with maxilla were extracted from 12 patients (8 males, 4 females) between 6 - 9 years old without medical history. Real-time polymerase chain reaction (PCR) was conducted to compare characterization of odontoblast cell in the 3rd and 10th passage, and between with bone inducing additive group and without additive group. Genes for odontoblasts characteristics are osteonectin (ONT), alkaline phosphatase (ALP), osteocalcin (OCN), dentin matrix protein 1 (DMP-1) and dentin sialophosphoprotein (DSPP). The level of gene expression was in a decreasing order of ONT, ALP, OCN, DMP-1 and DSPP in the 3rd passage, and in decreasing order of ONT, DMP-1, OCN, ALP, and DSPP in the 10th passage in the undifferentiation and differentiation group. The order of ONT, DMP-1, and OCN did not changed. ALP and DMP-1 were switched in order. ALP and DMP-1 may be used as important markers for differentiating between the 3rd passage and 10th passage cells. Considering that supernumerary tooth was extracted young age and the time required to cultured 10th passage was short, supernumerary tooth can be considered a useful donor site of dental pulp stem cells.

• Journal of the korean academy of Pediatric Dentistry
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• v.46 no.2
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• pp.219-225
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• 2019

The purpose of this study was to investigate the odontoblast gene expression related to the subculture speed of supernumerary dental pulp stem cells (sDPSCs). The stem cell is undifferentiated cells which has the ability to differentiate into various cells. Specific stimulation or environment induces cell differentiation, and these differentiation leads to bone or muscle formation. 20 sDPSCs were obtained from 20 children under aseptic condition. During the culture through the 10th passage, the third passage cells which showed short subculture period and 10th passage cells which showed long subculture period were earned. Each cell was divided into differentiated group and non-differentiated group. Quantitative real-time polychain reaction (q-RT-PCR) was performed for each group. The genes related to odontoblast differentiation, Alkaline Phosphatase (ALP), Osteocalcin (OCN), Osteonectin (ONT), Dentin sialophosphoprotein (DSPP) and Dentin matrix acidic phosphoprotein 1 (DMP-1), were measured. Differentiated cells showed more gene expression levels. Undifferentiated cells showed higher gene expression level in 10th passages but differentiated cells showed higher gene expression level in 3rd passages. Cells that showed faster subculture period showed relatively lower gene expression level except for OCN and DSPP.

• KOREAN POULTRY JOURNAL
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• v.41 no.11
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• pp.162-165
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• 2009

• Journal of the korean academy of Pediatric Dentistry
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• v.43 no.4
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• pp.419-426
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• 2016

For this research 20 supernumerary teeth impacted in the maxillary anterior have been extracted and pulp cells have been collected from them. From the collected pulp cells, total of 17 (10 males, 7 females) have been selected as subjects. From this research, the run-time of successive culture of the cell from tooth number pulp tissue was $2.91{\pm}0.29$ days. From the gathering of cells from the initial pulp tissue until gaining 80% confluency took $4.53{\pm}0.94$ which was the longest. The following successive cultures took $2.73{\pm}0.32$ days. Average runtime for female was $2.81{\pm}0.27$ days whereas male had average runtime of $2.98{\pm}0.29$ days. Average run-time for inversion was $2.94{\pm}0.30$ days and for normal location, $2.80{\pm}0.20$ days. Average runtime was $2.92{\pm}0.31$ days and other forms took $2.88{\pm}0.22$ days. In the future, follow up research would be needed to evaluate the efficiency of the cells collected from the initial passage and the latter passage as stem-cells and taking into consideration the less than 3 days'time for the subculture, it could be concluded that the research efficiency and fast cultivation would be sufficiently effective.

• Parasites, Hosts and Diseases
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• v.36 no.3
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• pp.191-198
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• 1998

The responses to antifolales of ToxopLasmc Bondii were investigated by measuring the dihydrorolate redLlctase (DHFR) activity. quantity of DHFR mRNA, and single-strand conformational polymorphism (SSCP) pattern. Pyrimethamine (PYM) and methotrexate (MTX) were tested ds anlifolates. When T. gondii was treated wish PYM, the viability was decreased by the increasing concentration of PYM. DHFR activity tended to increase as the passage proceeded. and the quantity of mRNA expressed was also increased according to passages. The viability of 7. gonnii was decreased by the increasing concentration of MTX, but it was maintained over 40% up to $100{\;}{\mu\textrm{m}}$ MTX. DHFR activity was 77.4% in the 1st passage ($1{\;}{\mu\textrm{m}}$). 82.2% in the 4th passage ($10{\;}{\mu\textrm{m}}$), and 141.3% in the 7th passage ($100{\;}{\mu\textrm{m}}$) But no changes were detected in SSCP pattern of T gondii rxposvd to FYM and MTX. both. These results suggested that the response of T gondii to FYN was rofulalcd by transrriptional level and that, in MTX. the viability of T. gonnii was derived from increasing DHFK activity.

• Current Research on Agriculture and Life Sciences
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• v.9
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• pp.21-28
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• 1991

The effect of subculture intervals and passages on plant regeneration from seed-derived callus was determined. Regeneration capacity of callus varied with rice cultivars and subculture intervars tested. The callus subcultured every 2 weeks produced more plants than that of 4 weeks. The calli from a Tongil-type rice cultivar, Milyang 23, lost easily their regeneration ability when the calli were subcultured every 2 weeks and 4 weeks. The callus induced from a japonica cultivar, "Yeongdeogbyeo", showed to maintain high frequency(>70%) of plant regeneration when it was subcultured every 2-week intervals. Casein hydrolysate supplemented in callus induction medium enhanced callus growth and its regeneration. High frequency of plant regeneration was obtained from the calli transferred on $N_6$ medium supplemented with kinetin(2mg/1) and NAA(1mg/1). The subcultured calli in the medium supplemented with casein hydrolysate(2 g/1), myo-inositol(200mg/1) and thiamine-HCl(2mg/1) increased the frequency of embryogenic callus formation and plant regeneration.