Background : Ginseng rain cover farming is expanding around Jinan county and Jangsu country of North Jeolla Province. Some farmers doing ginseng rain cover farming have suffered from difficulties due to hot weather damages. However, it is a situation that the study on mitigation techniques for high temperature damage do not exist with ginseng rain cover farming. Methods and Results : The test covering work was firstly done on April 28th for heat block film+90% black light blocking net, blue double sided film, and PE film+75% black light blocking net and when it comes to second treatment, 30% and 40% shading were implemented for heat block film group and blue double sided film group respectively and 75% black light blocking net was installed on PE film+75% shading group. When it comes to micro-climate measurement in rain cover facility, temperature, humidity and light intensity were measured during the growing period of ginseng. The results are as follows. Regarding the light transmittance (per PAR, 10 am in clear day) in facility with 1st covering, light block film covered group (LBF), blue double sided film group (BDF) and PE film group have 12.9±1.8%, has 11.6±1.0% and 27.1±1.1% respectively and after 2nd covering, in LBF groups, 30% blocking, 40% blcoking and no blocking have 10.6±1.3%, 8.2±0.9% and 12.9±1.8% and in BDF groups, 30% blocking, 40% blcoking and no blocking have 9.4±0.8%, 7.9±0.7% and 11.6±1.0 respectively and PE film group has 10.6±0.7%. Relative humidity also showed the same trend as temperature. The average monthly amount of light and maximum light intensity were lower in 30% and 40% light blocking groups of LBF and BDF and a little higher in no light blocking group compared to PE film group. The degree of high temperature damage was 1 in no LBF of BDF, but no LBF of LBF was so bad like 3. However, there was no high temperature damage in the test groups of blocking films or BDF with 30% and 40% light blocked light screens. Regarding root weight, all secondly treated groups of LBF group and BDF group were lighter compared to 4.36g of PE film group and especially, prism sheetof no light blocking group has 2.5g and BDF of of no light blocking group has 3.21g. 30 % and 40% light blocking groups of LBF group and BDF group were light with 3.20~4.07g. Conclusion : Regarding the analysis result on micro-climate in facility with different cover materials for 2 years old ginseng in ginseng rain cover farming of Gyeonggi Province, the covering method suitable for high temperature damage mitigation in ginseng rain cover farming was that 1st covering was done by PE film+75% black light blocking net and then 75% black light blocking net is additionally covered at a time when outside temperatures reaches 30℃.

Background : Ginseng, one of most famous traditional oriental medicines, has been known for a number of pharmacological properties including anti-tumor, anti-diabetic, anti-fatigue, anti-stress, anti-oxidative, and anti-aging effects. 20(S)-Protopanaxadiol (PPD), a intestinal metabolite of ginsenosides, is one of the active ingredients in ginseng. In this study, we investigated the synergistic anticancer effect of 20(S)-PPD and temozolomide (TMZ) and the mechanism of 20(S)-PPD on glioblastoma cells. Methods and Results : We examined cell viability and the morphological changes of C6 cells after treatment of 20(S)-PPD and TMZ. 20(S)-PPD showed a potent antiproliferative activity against C6 cells by triggering apoptosis. 20(S)-PPD-induced apoptosis was characterized by a dose-dependent mitochondrial damage. 20(S)-PPD and TMZ had a synergistic effect in increasing mitochondrial damage via caspase 3 activation. Conclusion : These results revealed an unexpected mechanism of 20(S)-PPD and TMZ, triggering a mitochondrial-mediated apoptosis in C6 cells. Our findings encourage further studies of 20(S)-PPD as a promising chemopreventive agent against glioblastoma.

Background : Recently, ginseng (Panax ginseng C.A. Meyer.) berry has been used as a health-promoting supplements. Also, Mulberries (Morus alba L.) fruit have been used in traditional herbal medicine to treat and prevent diabetes. In this study, we measured the cytotoxicity after fermentation of the extracts in Panax Ginseng Berry and Mulberry Fruit. Methods and Results : The extracts were prepared by decoction for 3 hours in distilled water (100 g/L). The dried extract was then dissolved in phosphate-buffered saline (PBS) in preparation for use. Cell viability was examined by an MTT assay. RAW 264.7 cells were seeded at 1 × 104/mL densities in 96-well plates. Each grouping had a non-treated group as the control. The extracts were added to each well and incubated for 24 hours at 37°C and 5% CO2. The MTT solutions (5 mg/mL) were added to each well, and the cells were cultured for another 2 hours. The supernatant was then discarded, and 100 μL of dimethyl sulfoxide was added to each well. The optical density was read at 540 nm. Conclusion : Probiotics and prebiotics modulate the composition of human and domestic animal gut microbiota. The beneficial effects may result from suppression of harmful microorganisms or stimulation of organisms which contribute in a positive way to the nutrition and health of human and domestic animal. Recently, fermentation using microorganisms for the production of more effective compounds has been extensively studied. In particular, the novel pharmacological effects of a new compound generated by fermentation have been reported. Some previous studies have demonstrated that Fermented herbal medicine extract showed better bioactivity than normal herbal Plants extract when used at the same concentration.

Background : Angelica dahurica Bentham et Hooker, Angelica gigas Nakai, Ostericum koreanum Maximowicz and Peucedanum japonicum Thumberg　are a major medicinal plant in north Geungbuk province. Using medicinal plants are impotant it`s ingredient. Dry condition and stroage method are not standard manual. The ingredient variation of dry condition and stroage method were not researched. Methods and Results : Using plant material were cutivated on Gyongsangbukdo Bonghwa area. It were studied ingredient variation after dry and storage condition by HPLC methods. Major ingredient of Angelica gigas Nakai are decurusin, decurusinangelate. Heated air bulk dry get more decursin than natuarl dry and decurusinangelate of natural bulk dry was higher than heated air bulk dry. Major ingredient of Ostericum koreanum Maximowicz are imperatorin and isoimperatorin.. Imperatorin of Ostericum koreanum was highest peak on 50℃ heated-air dry after plastic bag sorage and isoimperatorin was highest peak on 40℃ heated-air dry after mountain cultivation. Imperatorin is a major ingredient Angelica dahurica Bentham et Hooker. Heated air bulk dry get more decursin and decursinangelate than natuarl dry and small heated-air dry. Peucedanol-7o_glucoside is a major ingredient Peucedanum japonicum Thumberg. Natural bulk dry get more peucedanol-7o_glucoside than heated-air bulk dry. Conclusion : Ingredient of Angelica dahurica Bentham et Hooker, Angelica gigas Nakai, Ostericum koreanum Maximowicz are different under various cutivation, drying method, storage. Diffent Ingedients of Angelica gigas Nakai, Ostericum koreanum Maximowicz were not accord it’s optical conditon.

Background : The research is designed to investigate the optimal cultivation technology and the growth of above-ground and below-ground sections as well as the photosynthetic characteristics for new ginseng variety “K-1” by differentiating the planting density under the conditions of transplanting and direct seedling. Methods and Results : The K-1 variety and hybrid variety (Jakyungjong) were selected for the research and the ginseng varieties were transplanted and directly sown in Yeoncheon area in 2013. The transplanting was made in the form of 5 lines × 9 rows (45 plants), 6 lines x 9 rows (54 plants), 7 lines × 9 rows (63 plants) and 8 lines × 9 rows (72 plants) in each lot (1.65㎡) while the direct seedling for testing was conducted three times in randomly blocked design in the form of 11 lines × 14 rows (154 plants), 12 lines × 14 rows (168 plants), 13 lines × 14 rows (182 plants). Various measures were collected from the 4-year transplanted ginseng and 3-year direct seedling ginseng in 2015 to find out the growth features and photosynthesis of above-ground section (rate of germination, leaf length, leaf width, stem length and leaf area index (LAI)) and the below-ground section (length, diameter, weight and class of roots). Conclusion : After the planting of the ginseng, the germination rate of K-1 for the transplanting was 85.1 ~ 92.0% across different plantation densities while that for the direct seedling was 67.7% ~ 77.9% across plantation densities, thus showing no significant difference between the two planting methods. LAI was higher for the higher planation density for both transplanting and direct seedling. As for the photosynthesis speed, the form of 6 lines × 9 rows showed the higher speed in transplanting while the form of 12 lines x 14 rows showed the higher speed in direct seedling. The photosynthesis of K-1 was higher than that of Jakyungjong. In the 4-year ginseng cultivated under the transplanting, diameter of roots, number of branch roots and weight of raw ginseng were the highest in the plantation density of 5 lines × 9 rows. The distribution of root weight was high with 23.3% and 20.0% for the 51~70g group and the 71g or above group, respectively, for the 4 year transplanted plants in the form of 5 lines × 9 rows. The growth for above-ground and below-ground sections for K-1 was better than that for Jakyungjong. As a result, it was found that the proper plantation density for the 4-year root in the transplanted K-1 was 5 lines × 9 rows considering the growth of the above-ground section, quantity and distribution of root weight.

Hepatocytes and hepatic progenitors derived from human ES cells may be a useful source for clinical application. Therefore, identification and purification of these cell types would be following important issues. There are very few candidate surface markers that can be used to identify and purify hepatic progenitor cells. In addition, indocyanine-green can be uptaken by mature hepatocytes, but cannot be applied for fluorescence activated cell sorting (FACS) due to its long emission wavelength. In the present study, we tested EpCAM as a potential marker for magnetic-activated cell sorting (MACS) of hepatic progenitors and also modified indocyanine-green into fluorescent indomonocarbocyanine for FACS-mediated sorting of mature hepatocytes after differentiation of human ES cells. Hepatic progenitor cells were sorted by MACS after incubation with anti-human EpCAM antibodies. After the final differentiation, the differentiated cells and mouse primary hepatocytes (control group) were incubated with indomonocarbocyanine and were sorted by FACS. MACS and immunocytochemistry data showed that approximately 45% of differentiated cells were EpCAM-positive cells. EpCAM-positive cells expressed α-fetoprotein, FOXa2, HnF4a, and CK18. Differentiation efficiency into albumin-positive cells was significantly higher in EpCAM-positive cells, compared to EpCAM-negative cells. Importantly, indomonocarbocyanine successfully stained cells that expressed ALB. Furthermore, FACS analysis data showed that the purity of hepatocytes that expressed albumin was significantly increased after purification of indomonocarbocyanine-positive cells. Our data demonstrated that human ES cell-derived hepatic progenitors can be efficiently isolated by MACS using EpCAM antibody. In addition, we also showed that indomonocarbocyanine can be successfully used to identify and purify mature hepatocytes using FACS.

Highly homogeneous and functional stem cell-derived hepatocyte-like cells (HLCs) are considered a promising option in the treatment of liver disease and the development of effective in vitro toxicity screening tool. However, the purity of cells and expression and/or activity of drug metabolizing enzymes in stem cell-derived HLCs are usually too low to be useful for clinical or in vitro applications. Here, we describe a highly optimized differentiation protocol, which produces more than 90% albumin-positive HLCs with no purification process. In addition, we show that hepatic enzyme gene expressions and activities were significantly improved by generating three-dimensional (3D) spheroidal aggregate of HLCs. The 3D differentiation method increased expressions of nuclear receptors that regulate the proper expression of key hepatic enzymes. Furthermore, a significantly increased hepatic functions such as albumin and urea secretion were observed in 3D hepatic spheroids and HLCs in the spheroid exhibited morphological and ultrastructural features of normal hepatocytes. Importantly, we show that repeated exposures to xenobiotics facilitated the functional maturation of HLC, as confirmed by increased expression of genes for drug metabolizing enzymes and transcription factors. In conclusion, the 3D culture system with repeated exposures to xenobiotics may be a new strategy for enhancing hepatic maturation of stem cell-derived HLCs as a cell source for in vitro high-throughput hepatotoxicity models.

Hepatocyte-like cells (HLCs) derived from human pluripotent stem cells have received extensive attention in the development of drug screening and toxicity testing. However, it has been reported that stem cell-derived HLCs showed hepatic functions that were too limited to be of use in drug screening and toxicity testing, possibly due to the lack of sufficient intercellular communication under conventional two-dimensional (2D) culture conditions. Therefore, a 3D differentiation system may overcome the in vitro limitation of 2D culture to produce stem cell-derived hepatocytes with mature metabolic functions. In this study, the feasibility of using a silicone-based spherofilm, specifically designed to produce spherical cell clusters, to generate uniformly sized 3D hepatic spheroids from hESCs was investigated. Hepatic spheroids generated on the spherofilm showed more homogenous size and shape than those generated in conventional low-attachment suspension culture dishes. Results of immunohistochemical analysis showed that expression of the mature hepatic marker albumin (ALB) increased over time during the hepatic maturation process. Furthermore, the 3D culture system mimicked the in vivo 3D microenvironment. Laminin, which is an important component of hepatic ECM, was expressed in hepatic spheroids. The results of immunohistochemical analysis indicated that the 3D culture environment is capable of generating an in vivo-like microenvironment. In addition, quantitative PCR analysis showed that the mature hepatic marker ALB and cytochrome P450 (CYP) enzymes CYP3A4 and CYP3A7 were expressed at higher levels in 3D culture than in 2D culture. This indicates that the 3D culture system is suitable for hepatic maturation and that our size-controlled 3D culture conditions might accelerate hepatic function. These results suggest that 3D hepatic spheroids significantly enhance metabolic maturation of hepatocytes derived from hESCs

전라남도 연안에서 대량 생산되고 있는 해조류 5종(다시마․미역․김․파래․톳)을 대상으로 새로운 추출방법인 아임계 추출법을 도입하여 기존의 추출방법(열수, 용매추출)과 비교를 통하여 전남산 해조류의 기능성 측정 및 효율적인 추출물에 대한 자료를 확보하기 위해 조사를 실시하였다. 해조류 추출물 제조는 열수(80℃, 4시간), 용매추출(methanol, ethanol, 실온, 4시간)과 아임계 추출(압력 3 MPa, 온도 90, 150, 210℃)을 이용하여 제조하였다. 추출 수율은 아임계 추출＞열수 추출＞용매 추출 순으로 높게 나타났으며, 아임계 추출의 경우 추출 온도 조건이 높아지면서 수율 또한 증가하였으며 210℃에서 가장 높은 수율 결과를 나타냈다. 해조류 추출물의 총당 분석 결과 열수와 용매 추출의 경우 모든 시료에서 열수 추출이 높은 총당 함량을 보였으며, 아임계 추출의 경우 모든 조건(90, 150, 210℃)의 추출물이 용매 추출보다는 높은 총당 함량을 보였다. 시료별 총당 함량의 경우 열수 추출물은 파래, 김, 톳, 미역, 다시마 순으로 당 함량이 높았으며, 이러한 결과는 아임계 추출의 경우에서도 동일한 결과를 보였다. 환원당 분석 결과도 총당 결과와 유사하였다. 5종 해조류 추출물의 총 폴리페놀 함량은 210℃ 아임계 추출물＞150℃ 아임계 추출물＞90℃ 아임계 추출물＞열수 추출물＞methanol 추출물＞ethanol 추출물 순으로 높게 나타났다. 해조류 추출물의 DPPH radical 소거능을 측정한 결과, 모든 해조류 시료에서 열수 추출, 용매 추출, 3 MPa, 90℃ 아임계 추출물의 SC50 값은 서로 유사한 결과를 보였으며, 150℃ 아임계 추출물의 경우 농도가 조금 낮아졌으며, 특히 210℃아임계 추출물의 경우 SC50 농도가 현저히 낮아져 항산화 활성이 크게 증가하는 것으로 나타났다. 미백효과 실험을 위해 tyrosinase 저해 효과를 살펴본 결과 톳과 김이 가장 효과가 좋았으며 미역, 다시마, 미역귀 순으로 나타났고, 파래의 경우 거의 효과가 없는 것으로 나타났다. 결론적으로 새로운 추출방법인 아임계 추출법은 다른 방법에 비하여 높은 항산화 활성을 보여준다는 사실을 확인할 수 있었고, 이러한 추출공정은 식품소재로부터 기능성 성분을 보다 더 효과적으로 추출하는 방법으로 적용할 수 있을 것으로 판단된다.

MFG-E8 (Milk fat globule-epidermal growth factor VIII), also called lactadherin or BA46, SED1 is a glycoprotein found in milk and mammary epithelial cells, it is a major protein component associated with milk fat globule membrane. Previously, our study showed that expression of MFG-E8 is gradually increased with hepatic differentiation of human embryonic stem cells (hESCs). Therefore, we hypothesized that MFG-E8 would be an early cancer stem cell marker, which may predict cancer progression. Our results showed that MFG-E8 was expressed in various human cancer cell lines such as HepG2, Hep3B, and Huh7. Production and secretion of the MFG-E8 were also confirmed in the conditioned media of those three cell lines using enzyme-linked immunosorbent assay. Next, we analyzed the MFG-E8 expression in 11 clinical cases of cholangiocellular carcinoma (CC) and 33 cases of hepatocellular carcinoma (HCC) by immunohistochemistry and examined the potential correlation with β-catenin and AFP, which are known cancer markers. According to hitological criteria, the progression of HCC and CC was evaluated and classified into high, low, metastatic, and well-, moderate-, poor-differentiated, respectively. Statistical analysis indicated that incidence of both HCC and CC is significantly associated with male compared to female (P<0.05). Tumor size also has positive correlation with age (r2=08948). Our immunohistochemistry data showed that MFG-E8 was expressed both HCC and CC tissue. Interestingly, the MFG-E8 expression was significantly increased with cancer progression (P<0.05) in both cases. Additionally, b-cateninexpression was increased and its localization was changed from membrane to cytoplasm and nucleus with the degree of HCC. Likely b-catenin, AFP was also increased with the degree of HCC but it was not correlated with severalty of CC. Importantly, both AFP and b-catenin were highly co-localized with MFG-E8 in HCC. These results suggest that MFG-E8 may have important physiological roles and its expression in HCC and CC would be considered as an important prognostic factor.

The quality characteristics of commercial fermented vinegars made with different ingredients were compared. The pH levels of the persimmon, fig, and brewing and rice vinegars were 3.60, 3.37, and 2.62, respectively. The total acid contents of the brewing, apple, and plum vinegars ranged from 6.33 to 6.57%. The free amino acid contents were detected in the following order: brewing vinegar (521.05 mg/100 g) > fig vinegar (358.89 mg/100 g) > persimmon vinegar (353.02 mg/100 g) > rice vinegar (122.31 mg/100 g) > plum vinegar (103.52 mg/100 g). The free amino acid contents of the commercial fermented vinegars were 56.85~358.89 mg/100 g, and their gamma-aminobutyric acid (GABA) contents, 0.21~27.22 mg/100 g. In particular, the GABA content of the fig vinegar was 1.3- to 100- fold higher than those of the other vinegars. The total polyphenol compound and total flavonoid contents were detected in the following order: persimmon vinegar > fig vinegar > brewing vinegar > rice vinegar. Hence, the results of this study can provide a new alternative for making functional vinegars containing organic acid and GABA.

The purpose of this research is to distinguish the quantitative determination of phytochemicals in various agricultural products and to optimize an HPLC method for the determination of lycopene, lutein, α-carotene, β-carotene, and cryptoxanthin. Among the different conditions studied, the most suitable ones for our samples were the extraction with hexane/acetone/ethanol (50:25:25, v/v/v), dissolution of the dry extract in tetrahydrofuran/acetonitrile/methanol (15:30:55, v/v/v), injection on a C18 column with methanol/acetonitrile (90:10, v/v) + triethylamine 9 μM as mobile phase, and λdetection=475 nm. The mean percent recovery for the HPLC method were 120.7±4.1% (lycopene), 89.2±3.5% (lutein), 91.2±2.9% (α-carotene), 99.1±4.4% (β-carotene), and 100.0±5.3% (cryptoxanthin). The contents of lutein in the agricultural products were spinach, kiwi, tomato, blueberry, melon, respectively. However, the lycopene contents were the highest in the Black tomato (56.66±7.48 mg/kg) and Jangseong tomato (50.28±5.42 mg/kg). The concentration of β-carotene in all of the agricultural products ranged from 0.07 mg/kg to 65.03 mg/kg. The quercetin content of the agricultural products increased in the order of blueberry (986.57∼1,054.06 mg/100 g), kiwi (44.96∼55.09 mg/100 g), hallabong (31.92∼35.60 mg/100 g), and tomato (26.38∼34.94 mg/100 g). The highest kaempferol content was found in the blueberry (47.79∼76.15 mg/100 g) with results in all of the tested samples varying between 6.54∼48.11 mg/100 g. The total polyphenol contents of the various agricultural products increased in the blueberry (213.60∼229.96 mg/100 g), spinach (112.50∼141.67 mg/100 g) and kiwi (46.49∼70.44 mg/100 g). The total flavonoid content was the highest in both blueberry and spinach. Vitamin C content was detected in kiwi > hallabong > tomato > blueberry, respectively. The total anthocyanin contents (TAC) was detected in the Damyang blueberry and the imported blueberry.

In this study, the proximate compositions, minerals, free amino acids, total polyphenol and flavonoids of figs were analyzed according to their cultivars. The proximate compositions showed moisture at 85.37∼87.28%, crude ash at 0.38∼0.43%, crude lipid at 0.20∼0.26%, crude protein at 0.39∼0.81%, carbohydrate at 11.23∼13.66% and crude fiber at 5.12∼7.55% (dry base). The amount of the following minerals in the figs were highest, in this order: K 〉Ca 〉Mg 〉Na. The total polyphenol contents were highest in this order: Dauphine (198.91∼261.64 mg/kg), Banane (211.07 mg/kg) and Horaish (169.90∼174.33 mg/kg). The total flavonoid contents were highest in this order: Banane (84.52 mg/kg) 〉Dauphine (60.47∼68.71 mg/kg) 〉Horaish (44.12∼44.60 mg/kg). The quercetin contents were highest in this order: Dauphine, 2.40∼3.54 mg/kg; Banane, 3.54 mg/kg; and Horaish, 2.40∼2.75 mg/kg; but the flavonoid contents were lowest in this order: Dauphine, 1.11∼1.16 mg/kg; Banane, 1.14 mg/kg; and Horaish, 1.09∼1.11 mg/kg. The free amino acid content of the figs was 199.70∼328.77 mg/100 g; their essential free amino acid contents, 46.45∼67.46 mg/100 g; and their GABA (γ-aminobutyric acid) contents, 13.57∼26.69 mg/100 g.

Compared to wide ranges of genetic variation of natural populations, very limited Miscanthus cultivar has been released. This study was the first report on the development of Miscanthus cultivar by means of radiation breeding. Seeds of M. sinensis were initially exposed to gamma rays of 250 Gy for 24 h, generated from a 60Co gamma-irradiator. The irradiated seeds were sown and then the highly tiller-producing mutants were selected for this study. Biomass-related parameters including tiller number, plant height, stem diameter, and leaf number were measured. Ploidy level and internal transcribed spacer (ITS) were investigated to characterize the mutants compared to wild type (WT) Miscanthus. Plant height and tiller number were negatively related, where multi-tillering mutants were relatively short after 4 month growth. However stem diameter and leaf number were greater in mutants. All the materials used in this study were diploid, implying that the mutants with greater tiller numbers and stem diameter were not likely related to polyploidization. Based on the sequence of ITS regions, the mutants demonstrated base changes from the gamma irradiation where G+C content (%) was decreased in the ITS1, but increased in ITS2 when compared to WT sequence. ITS2 region was more variable than in ITS1 in the mutants, which collectively allows identification of the mutants from WT. Those mutants having enhanced tillers and allelic variations might be used as breeding materials for enhanced biomass-producing Miscanthus cultivars.

Understanding the host defense mechanisms in response to brown leaf spot disease caused by Cochliobolus miyabeanus is very important for production of resistant plant. In this study, two-dimensional gel electrophoresis (2-DGE) in conjunction with mass spectrometry was utilized to unravel changes of stress inducible proteins in rice leaves infected with C. miyabeanus. For this purpose, we firstly observed disease developmental process of C. miyabeanus in rice using trypan blue, anilin blue, acid fuchsin staining, and DAB staining for ROS detection and expressional abundance of ROS related proteins in rice leaves inoculated was confirmed by Western blotting. Proteins were extracted by PEG fractionation and their expression patterns were analyzed by 2-DGE and subjected to image analysis using the ImageMaster 6.0 2D Platinum software, resulting in the identification of 86 differentially expressed protein spots with significantly changes (p<0.05) compared with control. MALDI-TOFTOF-MS analysis revealed that 69 proteins including 42 and 27 significantly up- and down-regulated proteins, respectively, were identified. Based on gene ontology analysis, identified proteins were classified according to their functional groups: metabolism (20%), oxygen-detoxifying (13%), protein stress/defense (24%). Thus, these results for the first time suggest that differentially induced proteins may play a key role for understanding host defense mechanisms during rice -C. miyabeanus interaction.

Mesenchymal stem cells (MSC) are of great interest for cell-based therapies and tissue engineering approaches, as these cells are capable for extensive self-renewal and display a multilineage differentiation potential. Clinical application of these cells for degenerative and age-related diseases has been accumulating. However, preparation of MSC before the onset of the diseases, it needs to develop the cryopreservation method. Most cryopreservation methods include fetal bovine serum (FBS) which is essential for effective cryopreservation. Yet it should not be used clinically because of the potential risk of infection. In the present study, we investigated whether human serum albumin (HSA), human serum (HS), and knockout serum replacement (KSR) can be used as an alternative of FBS for cryopreservation of human adipose derived stem cells (hADSC). Cells cryopreserved with 9% HSA showed much higher viability after thawing compared with cells frozen with 5% or 1% HSA. Cells cryopreserved with 90% HS or KSR exhibited greater viability than cells frozen with 25% and 5% HS or KSR, respectively. Viability of cells frozen with 9% HSA, 90% HS or 90% KSR was comparable to that with 90% FBS. Morphology and proliferation ability of these cells were not affected by cryopreservation when compared the freshly obtained cells. Cryopreserved hADSC expressed transcription factor genes including Oct3/4, Nanog, Nestin and Sox2, which are related to the self-renewal of stem cells. Flow cytometric analyses showed that both fresh and cryopreserved hADSC were positive for the antigens of HLA-ABC, CD44, CD73, CD90, and CD105, CD166, and negative for HLA-DR, CD31, and CD34. Similar to fresh cells, cryopreserved hADSC could differentiate into mesodermal lineages, adipogenic, osteogenic, or chondrogenic cells. These results suggest that 9% HSA, 90% HS or 90% KSR can be used to replace FBS during successful cryopreservation of hADSC.

The human eyelid adipose-derived stem cells (HEACs) are known as a candidate source for stem cell-based therapy. HEACs possess the ability to proliferate in vitro and multipotency to differentiate into adipogenic, osteogenic and chondrogenic cells. To be used later than the time of collection, a long-term storage is needed. In this study, we investigated stem cell characteristics after cryopreservation of HEACs for 6 months and 1 year in liquid nitrogen. Frozen-thawed stem cells have shown that cumulative cell and doubling numbers were similar to those of fresh HEACs. After thawing, HEACs expressed stem cell-related genes of SCF, NANOG, OCT4, and TERT, ectoderm-related genes of NCAM and FGF5, mesoderm/endoderm-related genes of CK18 and VIM. They also consistently expressed transcripts of the immune-related genes of HLA-ABC and β2M. To induce mesodermal differentiation, cell were cultivated in adipogenic, osteogenic or chondrogenic medium for 2～3 weeks. After each differentiation culture, HEACs expressed adipocyte-, osteocyte- and chondrocytespecific genes. They were also stained with Oil red O, von Kossa, or alcian blue, revealing adipogenic, osteogenic, or chondrogenic character, respectively. The results suggest that long-term storage up to 1 year do not affect their biological properties, HEACs may be suitable for clinical application on cell-based therapies.