• 한국미생물·생명공학회지
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• 제17권3호
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• pp.231-235
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• 1989

부착성 동물세포인 recombinant human embryonic kidney cell line 293을 5% FBS가 함유된 DME/F12(3:1) mixture 배지에서 배양할 경우 static culture에서와는 달리 미립담체 배양에서는 낮은 부착효율과 spreading의 저해현상이 관찰되었다. Cell line 293의 미립담체 배양에 있어서 배지내의 serum은 세포의 미립담체에 대한 부착속도(attach-ment rate)를 감소시킬 분만 아니라 세포의 증식을 저해한다는 사실을 확인하였다. 이러한 경우, conditioned microcarrier를 이용함으로써 미립담체로부터 세포의 탈착을 어느 정도 막을 수 있었다. 특히 autocrine growth factor를 생성하는 hepatoma cell line인 HpG 2 cell을 배양하여 얻은 conditioned medium을 serum-free medium에 첨가하여 사용함으로써 세포의 정상적인 성장 분만 아니라 growth-stimulation을 유도할 수 있었다.

• International Journal of Oral Biology
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• 제35권1호
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• pp.1-5
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• 2010

Human fibroblasts that maintain the structural integrity of connective tissues by secreting precursors of the extracellular matrix are typically cultured with serum. However, there are potential disadvantages of the use of serum including unnatural interactions between the cells and the potential for exposure to animal pathogens. To prevent the possible influence of serum on fibroblast cultures, we devised a serum-free growth method and present in vitro data that demonstrate its suitability for growing porcine fetal fibroblasts. These cells were grown under four different culture conditions: no serum (negative control), 10% fetal bovine serum (FBS, positive control), 10% knockout serum replacement (KSR) and 20% KSR in the medium. The proliferation rates and viabilities of the cells were investigated by counting the number of cells and trypan blue staining, respectively. The 10% FBS group showed the largest increase in the total number of cells ($1.09\;{\times}\;10^5\;cells/ml$). In terms of the rate of viable cells, the results from the KSR supplementation groups (20% KSR:64.7%; 10% KSR: 80.6%) were similar to those from the 10% FBS group (68.5%). Moreover, supplementation with either 10% ($3.0\;{\times}\;10^4\;cells/ml$) or 20% KSR ($4.8\;{\times}\;10^4\;cells/ml$) produced similar cell growth rates. In conclusion, although KSR supplementation produces a lower cell proliferation rate than FBS, this growth condition is more effective for obtaining an appropriate number of viable porcine fetal fibroblasts in culture. Using KSR in fibroblast culture medium is thus a viable alternative to FBS.

• Biotechnology and Bioprocess Engineering:BBE
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• 제1권1호
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• pp.32-35
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• 1996

Amoving aeration-membrane (MAM) bioreactor was employed for the production of 2$\mu$g/mL of tissue type Plasminogen Activator (tPA)in serum free medium from normal human fibroblast cells. This system could maintain high cell density for long periods of steady state conditions in perfusion cultivation. Under normal operating condition, shear stress was as low as 0.65 dynes/$\textrm{cm}^2$ at the agitation speed of 80 rpm. Even though cell density gradually decreased with increasing agitation speed, tPA production increased linearly with increasing shear stress within a moderate range. This culture system allowed production of 2$\mu$g tPA/mL while maintaining a high cell denisty of 1.0$\times$107 viable cell/mL.

• 약학회지
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• 제43권1호
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• pp.85-90
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• 1999

FTY720, a novel immunosuppressant, elevated the level of endogenous sphingoid bases in a dose-dependent manner within 3 hr in $LLC-PK_1$ cells. The relative molar ratio of sphingoid bases expressed as sphingosine/sphinganine (SPN/SPA), a biomarker of altered sphingolipid biosynthesis, in $10{\;}{\mu}M$ of FTY720 showed tow-fold increase as compared with the one in control culture. FTY720 under the serum-free medium condition increased only cytosolic free sphingosine concentration, not sphinganine concentration in a time-dependent manner over the 8 hr incubation under the same condition as in serum free cultures, the SPN/SPA ratio began to fluctuate and the number of floating cells as an indicator of cytotoxicity was increased 8 hr after the addition of FTY720 to cultured cells. These results suggest that the process of FTY720-induced cell death in $LLC-PK_1$ cells.

• Korean Chemical Engineering Research
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• 제44권1호
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• pp.73-80
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• 2006

본 연구에서는 제대혈 유래의 조혈줄기세포를 효과적으로 배양하기 위한 선행 연구로서 세포 배양 환경에 따른 조혈줄기세포 증식능의 변화를 관찰하였다. 제대혈의 단핵구 세포에서 분리한 $CD34^+$ 세포를 성장인자 조성-I(이하, coc-I) (EPO, GM-CSF, SCF, IL-3) 및 성장인자 조성-II(이하, coc-II) (TPO, G-CSF, SCF, IL-6, Flt3/Flk-2 ligand)가 포함되어 있는 IMDM(Iscove's modified Dulbecco's medium) 및 무혈청 배지(serum free media, SFM)에서 배양하였으며, 우태아혈청(FBS)의 첨가 영향, 2차원 및 3차원 배양 후 각 조건에서의 세포 증식 및 콜로니 형성능을 비교하였다. 일반적으로 coc-I에서의 세포 증식 및 콜로니 증식이 coc-II에서보다 높았다. 3차원 배양(methocult)에서는 가장 높은 세포 증식($2,258{\pm}456$배)을 나타냈으며, 같은 조성의 2차원 배양(IMDM + coc-I + FBS)에서는 가장 높은 콜로니 증식(BFU-E: $652{\pm}19$, CFU-GM: $520{\pm}58$, CFU-GEMM: $339{\pm}100$배)이 나타났다. 배지를 기준으로 보면, coc-II 조성에 우태아혈청이 포함되지 않은 경우를 제외한 모든 경우에서 세포 증식 및 콜로니 증식이 무혈청 배지에서보다 IMDM에서 높았다. 결론적으로, 모든 배양 조건 중에서 'IMDM + coc-I + FBS' 및 'IMDM + coc-I'에서 가장 좋은 콜로니 증식을 보였으며, 우태아혈청의 첨가 및 2차원 배양 조건이 콜로니 증식에 더 효과적인 것으로 확인되었다. 본 연구 결과는 앞으로 조혈줄기세포의 체외 증식에 필요한 공정개발이나 생물반응기 설계에 유용한 정보를 제공할 수 있을 것으로 사료된다.

• 한국미생물·생명공학회지
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• 제22권4호
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• pp.360-367
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• 1994

The effect of various nutrients for the production of the anti-complementary polysaccha- ride by Flammulina velutipes, was examined and the partial purification of the polysaccharide was carried out. The culture conditions and medium compositions considerably influenced the anti-complementary activity of the polysaccharides. When a culture was carried out at 23$\circ$C and pH 5.5 for 6 days in the synthetic medium supplemented with galactose and NaNO$_{3}$, the production of the anti-complementary polysaccharide was maximized. The crude polysaccharide, FV-1 was isolated from the culture broth and partially purified into two fractions, FV-1-II and FV-1-III by gel filtration using Sephadex G-100. FV-1-II was mainly consisted of xylose, mannose, galactose and glucose, and rhamnose, mannose and galactose, for FV-1-III. Also, the anti-complementary activity of FV-1-III was reduced partially in the absence of the Ca ion. When crossed immunoelect- rophoresis using anti-human C3 serum was carried out after incubation of normal human serum with the FV-1-III in the Ca ion free condition, a cleavage of C3 precipitin line was observed. These results indicate that the mode of complement activation by polysaccharide from Flammulina velutipes is via not only the classical pathway but also the alternative pathway.

• 한국가축번식학회지
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• 제27권4호
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• pp.339-347
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• 2003

The purpose of this study was to investigate the development of bovine nuclear transfer (NT) embryos cultured in serum-free conditions. Bovine NT embryos cultured in various culture conditions were compared blastocyst development, total cell number and apoptosis using TUNEL assay. In experiment 1, blastocyst rates of NT embryos were significantly higher (P<0.01) in FBS (22.0％) and BSA (26.6％) groups than in PVA (6.3％) group. Total cell number was significantly higher in FBS (78.4$\pm$19.4) and BSA (90.9$\pm$29.1) groups than in PVA group (46.0$\pm$0.0). Apoptotic cell number was significantly fewer in FBS (3.1$\pm$1.4) and BSA (1.7$\pm$1.4) groups than in PVA group (7.0$\pm$20.0) However, all of results were not different between the FBS and BSA group. In experiment 2, blastocyst rates of NT embryos were significantly higher (P<0.05) in fatty acid free-BSA (FAF-BSA) group (26.8％) than in fraction V-BSA group (11.2％). Total cell number were somewhat higher in FAF-BSA group (89.8$\pm$30.7) than in fraction V-BSA group (88.1$\pm$19.3). Apoptotic cell number were somewhat fewer in FAF-BSA (1.7$\pm$1.5) group than in fraction V-BSA group (4.2$\pm$2.9). These findings suggest that serum free condition were effective for the in vitro development of bovine NT embryos. Therefore, we concluded that fatty acid free-BSA has beneficial effect in development bovine NT embryos and can be use as a serum substitute.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.71-71
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• 2003

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifunctional cytokine that has been implicated in the regulation of pre-implantation embryo development across several species. The aim of this study was to determine the effects of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) on development of porcine parthenotes and nuclear transferred embryos, and on their expression of implantation-related genes. In the presence of bovine serum albumin, mGM-CSF did not increase the percentage of oocytes that developed to the blastocyst stage and at day 7 did not increase oocyte cell number. Addition of 10 mM GM-CSF to protein-free culture medium significantly increased the compaction and blastocoel formation of 1- to 2-cell parthenotes and cloned embryos developing in vitro. However, cell number was not increased when they were cultured in the presence of GM-CSF. Semi-quantitative reverse transcripts polymerase chain reaction (RT-PCR) revealed that mGM-CSF enhances mRNA expression of the leukemia inhibitory factor receptor, but does not influence interleukin-6 or sodium/glucose co-transporter protein gene expression in blastocyst stage parthenotes. These results suggest that mGM-CSF may enhance viability of porcine embryos developing in vitro in a defined culture medium.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권3호
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• pp.254-261
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• 2005

The over-expression of Bcl-2 has greatly improved the culture period, specific growth rate, and maximum viable cell density of NS0 cells culture under low serum condition. Further analysis of these data suggests that a saturation model of the Monod type can be used to represent the relationships of specific growth rate and initial serum concentration. The ${\mu}_{max}$ and $K_s$ for the Bcl-2 cell line is $0.927day^{-1}\;and\;0.947\%(v/v)$ respectively, which are $21\%$ greate and $7\%$ lower respectively than its control counterpart. Study on the amino acid supplementation revealed that Bcl-2 cell lines possess greater improvement in the specific growth rate and maximum viable cell density compared to the control cell lines. A further increase in the amino acid supplementation has resulted a $17\%$ decrease in specific growth rate and no improvement in maximum viable cell density in the control culture. However, the Bcl-2 cell line exhibited a better growth characteristic in this culture condition compared to that of control cell lines. The higher specific growth rate and maximum viable cell density of the Bcl-2 cell line in medium fortified with serum and MEM EM suggested a more efficient nutrient metabolism compared to that in the control cell line. The low serum and amino acid utilisation rate and the higher cell yield may prove to be important in the development of serum/protein free culture.

• KSBB Journal
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• 제21권2호
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• pp.110-117
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• 2006

일반적인 세포주의 동결보존은 혈청이 첨가된 배양배지에 10% DMSO를 첨가하여 실행하게 된다. 그러나 무혈청 환경에서 배양되는 세포주를 이용하여 생물의약품을 생산하는 공정에 사용되는 세포주의 경우는 교차오염 방지를 위해 동결보존 역시 혈청을 제거한 상태에서 실시되어야 한다. 본 실험은 무혈청 동결보존이 CHO 세포에 어떠한 영향을 미치는지 알아보기 위하여 실시되었다. 우선, 무혈청 동결보존에서 세포 생존율을 높이기 위해 투과성 및 비투과성 첨가제를 배지에 첨가하는 방법으로 동결보존 및 해동하여 생존율을 측정하였다. 그 결과, 10% DMSO와 0.03 M raffinose를 동시에 첨가한 경우에 76%의 생존율을 확보할 수 있었지만 기존의 혈청을 이용하는 동결보존에는 미치지 못하였다. 두 번째로 무혈청 배지의 동결보존 능력을 알아보기 위해 시판 중인 무혈청 배지와 무혈청 동결보존제를 사용하여 동결 보존 후의 생존율을 비교한 결과, 무혈청 배지를 이용한 실험에서는 혈청 배지를 이용한 동결보존과 유사한 95% 이상의 생존율을 확인할 수 있었다. 이는 무혈청 동결보존제의 동결보존 능력보다 우수한 결과이다. 마지막으로 무혈청 배지를 이용한 장기간 동결보존에서 CHO 세포의 안정성을 확인하기 위하여 무혈청 배지로 동결보존된 CHO 세포를 3개월 단위로 해동하여 생존율 및 성장 회복율을 측정하고, real-time RT-PCR을 통해 삽입된 CHO DHFR 유전자의 안정성을 평가하였다. 혈청배지와 비교할 때, 생존율과 성장 회복율, 유전자 안정성 측면에서 모두 동일한 결과를 보여, 무혈청 배지를 이용한 18개월 이상의 동결보존이 안정적임을 검증할 수 있었다. 결과적으로 생물의약품 공정에서 무혈청 배지를 이용한 동결보존이 혈청을 이용한 동결보존을 대체할 수 있을 것으로 생각된다.