• KSBB Journal
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• 제8권1호
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• pp.28-35
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• 1993

The serum free medium was developed and used for the suspension culture of mammalian cells. Although there were the problems of the longer lag time and the smaller maximum cell concentration achievable, the higher specific productivity as well as other advantages of the serum free medium can make it a more realistic alternative. The existence of a staggering period in glucose concentration vs. time profile in the batch culture can be a practical indicating signal for performing fed batch culture. The concentration dependence of the effects of the additives in the serum free medium as well as its economic feasibility was also tested.

• 생명과학회지
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• 제24권12호
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• pp.1301-1307
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• 2014

지방유래줄기세포(adipose-derived stem cell; ADSC)는 조직재생을 위한 탁월한 수단으로 인정되고 있으나 세포증식속도가 느려 ADSC의 배양용 배지에는 대게 fetal bovine serum (FBS)이 첨가된다. FBS는 세포에 다양한 영양분을 공급하지만 세포의 기능에 영향을 미칠 수 있는 미 동정 물질도 많이 함유하고 있다. FBS에 의한 예상밖의 영향과 동물유래물질의 오염을 방지하기 위해 ADSC의 무혈청 배양법에 관한 연구가 광범위하게 이루어지고 있다. 본 연구에서는 ADSC세포에 SV40의 T항원 유전자를 도입하여 증식속도를 향상시킨 ADSC-T세포주의 효율적인 무혈청 배양법을 확립하기 위해 ADSC-T의 세포증식에 미치는 아미노산복합체, 비타민 복합체 및 여러가지 영양분 혼합물(B27)의 영향을 검토하였다. 그 결과, ADSC-T세포를 DMEM/F12 무혈청 배지에 현탁하여 plate에 주입하였을 때는 증식하지 않았으며 아미노산, 비타민 및 B27 영양소복합체는 증식촉진효과를 나타내지 않았다. 그러나 ADSC-T세포를 유혈청 DMEM배지로 24시간 배양 후 DMEM/F12 무혈청 배지로 교체하여 배양했을 때는 세포가 증식하였으며 이때, 비타민 복합체와 B27 영양소복합체는 증식촉진효과를 나타내었다. 또한 Stem pro 배지를 이용하면 ADSC-T의 무혈청 부유배양이 가능한 것으로 나타났다. ADSC-T세포는 분자량 70 kDa 부근의 단백질을 다량으로 분비하였으며, 성장인자 중에서 insulin-like growth factor (IGF)와 fibroblast growth factor basic (FGF basic)는 유혈청 배양보다 무혈청 배양에서 더 많이 분비되었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.171-173
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• 2003

본 연구에서는 생분해성 고분자 나노입자를 이용하여 부착성 세포의 세포군집 (cell aggregates) 형성을 촉진시켜 무혈청 배지에서 3차원적 부유 배양하는 방법을 개발했다. 생분해성 고분자 나노입자의 사용은 무혈청 배지 부유배양에서 부착성 동물세포인 HEK 293 세포의 세포군집 형성과 세포증식(나노입자를 사용하지 않은 대조군과 비교하여 2배 이상)을 촉진하였다. 일반적으로 무혈청배지 부유배양에 세포를 적응(adaptation)시키는 데에는 시간이 오래 걸리고 많은 비용이 드는데, 이 연구에서 개발된 방법은 이러한 세포적응 공정이 필요없다. 이 배양법은 여러 부착성 동물세포의 산업적 대량배양에 유용하게 사용될 수 있을 것이다.

• 한국수정란이식학회지
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• 제29권3호
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• pp.229-234
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• 2014

The aim of the present study was to compare two different serum-free media, modified synthetic oviduct fluid (mSOF) and modified potassium simplex optimization medium (mKSOM) containing 20% RD (RPMI1640 + DMEM, 1:1 v/v) (RD-mKSOM), for in vitro culture (IVC) of bovine embryos. After in vitro maturation and fertilization, the presumptive zygotes were cultured in two different serum-free conditions for 7 days and 9 days to evaluate blastocyst formation and hatching, respectively. Serum supplemented conventional CR2 medium was used as control. After 7 day of culture, there was no significant difference in cleavage and blastocyst formation rates among three groups (mSOF, 59.3 and 30.1%; RD-mKSOM, 65.0 and 41.5%; control, 51.6 and 38.0%, respectively). Hatching rate was significantly higher in control (69.0%) than other experimental groups (mSOF, 22.0%; RD-mKSOM, 39.5%) (P<0.0001 and P<0.001, respectively). Although both serum-free conditions showed lower hatching rates than serum-added control, in serum-free groups, RD-mKSOM showed significantly higher hatching rate than mSOF (P<0.001). In addition, one-step using RD-mKSOM may facilitate IVC procedure than two-step culture system. In conclusion, the results indicate that one-step RD-mKSOM is more suitable defined culture system for IVC of bovine embryos than two-step mSOF.

• 대한수의학회지
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• 제36권3호
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• pp.551-563
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• 1996

This study investigated the properties of primary cultured proximal tubule cells in hormonally defined(insulin, transferrin, and hydrocortisone), serum-free medium or 10% serum-supplemented medium. The growth rate of the primary cultured proximal tubule cells was lower in the hormonally defined, serum-free medium than in the 10% serum- supplemented medium(p < 0.05), while the activities of brush border marker enzymes, alkaline phosphatase(AP), leucine aminopeptidase(LAP), and y-glutamyl transpeptidase(${\gamma}$-GTP) were increased(p < 0.05). The activities of these enzymes, however, decreased with the lapse of incubation time to 50-70% after 6 days culture compared to those of the freshly-prepared proximal tubules. The enzymatic activities of the primary cultured proximal tubul cells on 6, 9, 12, and 15 days of culture were significantly increased in the hormonally defined, serum-free medium compared to the 10% serum-supplemented medium(p < 0.05). The functional differentiation of the primary culture was examined by observing multicellular domes of the confluent monolayer, which is indicative of transepithelial solute transport. The dome formation by the proximal tubule cultures occurred at a higher frequency in the hormonally defined, serum-free medium than in the 10% serum-supplemented medium(p < 0.05). Upon electron microscopic examination, an increased density of the brush border was observed in the hormonally defined, serum-free medium compared to the cells grown in 10% serum-supplemented medium. The activities of $Na^+$glucose cotransporter($^{14}C$-a-MG uptake), $Na^+$phosphate cotransportere($^{32}P$ uptake) and $Na^+$ transporter($^{22}Na^+$ uptake) in the brush border membrane, and of $Na^+/K^+$-ATPase($^{86}Rb$ uptake) in the basolateral membrane were significantly stimulated in the hormonally defined, serum-free medium than in 10% serum-supplemented medium(p < 0.05). In conclusion, the primary cultured proximal tubule cells grown in the hormonally defined, serum-free medium demonstrated a slower growth rate, but the functions of cell were enhanced.

• KSBB Journal
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• 제13권3호
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• pp.295-302
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• 1998

Human Erythropoietin (EPO) gene is cloned in quail fibrosarcoma cell, QT35. Because molecular weight of EPO is similar to that of serum albumin, cell culture with serum containing medium makes purification of EPO very difficult. Using fractional factorial study, we have developed serum free medium for the recombinant QT35 cell lines, QT N4D4 and QT SY-IMP, which have cloned EPO with glutamine synthetase (GS) gene amplification system and with puromycin selective marker, respectively. Among the seven frequently used medium components, fibronectin, BSA, and EGF were the most important for EPO production. However, sufficient fibronectin supplement to the medium did not make any good attachment of QT35 to culture plate over 3 days. Therefore, to maximize EPO production, we attempted a medium-shift at confluence from serum containing medium to serum free medium(QT SFM6). Using the medium-shift protocol with QT SFM6, nearly the same productivity of EPO was achieved comparing with that without medium-shift. This result was true in both QT35 cell lines in three types of culture, i.e. T flask, microcarrier and roller bottle cultures.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1299-1303
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• 2005

Human articular chondrocytes (HAC) were cultivated as a monolayer in a serum-free medium for primary culture (SFM-P). An optimized SFM-P provides $95\%$ proliferation rate of that obtainable from primary and secondary chondrocyte cultures grown in a control medium with serum. The gradual decrease in the amounts of synthesized glycosaminoglycan and type II collagen was improved by coating the culture dishes with type IV collagen and fibronectin. A significant improvement in the expression of type II collagen and aggrecan mRNA could be achieved. In addition, the monolayer cultures showed better synthesis of the extracellular matrices than alginate-bead cultures in SFM-P.

• 한국미생물·생명공학회지
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• 제17권5호
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• pp.489-493
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• 1989

쥐 하이브리도마 Alps 25-3를 이용하여 무혈청 배지 KM3의 각 성분이 세포증식과 항체생성에 미치는 영향을 조사하여 무혈청 배지를 최소화한 KMS를 결정하였다. Alps 25-3에 영향을 미치는 것으로 조사된 transferrin, ethanolamine 그리고 BSA만을 혼합하여 결정한 KMS를 다른 쥐 하이브리도마인 A4W, KW 그리고 HCGK에 적용시켰다. KM5를 KM3와 비교하였을 때 세포증식과 항체생산이 거의 동일한 양상을 보였다. 그러나 혈청배지와 비교하여 보면 최고 세포농도는 50-90%, 최종 단일클론항체의 농도는 85-100%의 수준을 보였다. 본 연구의 결과로 쥐 하이브리도마 세포를 무혈청 배지에서 배양시킬 때 필수적으로 첨가기어야 하는 성분들이 조사되었으므로 대량배양에 응용하거나 대사를 연구하는데 유용하게 이용되어지리라 본다.

• Journal of Animal Science and Technology
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• 제58권3호
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• pp.11.1-11.6
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• 2016

Background: This study was investigated the effects of culture media conditions on production of eggs fertilized in vitro of embryos from ovaries of high grade Korean native cow, Hanwoo. Methods: The IVMD 101 and IVF 100 were used for in vitro maturation of selected Hanwoo oocytes and In vitro embryo culture after in vitro fertilization, respectively. The IVMD 101 and IVD 101 were used for in vitro culture and completely free of serum. Results: The cleavage rates of 2-cell embryos in reference to Hanwoo oocytes were 86.7, 92.9, and 90.1 % in the control group, IVDM101 medium and IVD101 medium, respectively which indicates that the IVDM101 medium and IVD101 medium may result favorable outcomes. The in vitro development rates of blastocysts were 12.4, 38.4 and 32.4 % in the control group, serum free IVMD101 medium and IVD101 medium, respectively. For hatched blastocysts, it was 5.3, 33.9, and 28.6 % in the control group, serum free IVMD101 medium and IVD101 medium, respectively. Hence, more favorable results were expected for the hatched blastocysts in which the IVMD101 medium and IVD101 medium were used than the control group. Average cell numbers of blastocysts were 128.3, 165.7, and 163.6 in the groups of TCM-199 + 10 % FBS medium, IVMD 101 medium, and IVD 101 medium, respectively which clearly show that the IVMD 101 and IVD 101 medium consequence significantly higher cell numbers compared to the control group (i.e., TCM-199 +10 % FBS medium). Pregnancy rate after embryo transfer was 39.6 % when the serum free medium was used which is higher than that of the medium supplemented with serum (32.8 %). In addition, stillbirth rates were 4.9 % in the group of serum free medium whereas it was 13.6 % in the serum supplemented medium (13.6 %). Conclusions: Taken altogether, serum free media, the IVMD 101 and IVD 101 represented more favorable results in the embryo development rate of embryos, cell numbers of blastocyst, and pregnancy rate. Of note, the IVMD 101 medium showed better outcomes hence, it might be a better option for future applications for in vitro culture of bovine embryos.

• Asian-Australasian Journal of Animal Sciences
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• 제9권5호
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• pp.583-590
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• 1996

The purpose of this study was to evaluate some factors in the bovine embryonic development from one-cell to blastocyst using modified synthetic oviduct fluid medium (mSOFM), after maturation and in vitro fertilization of the oocytes. The embryonic development to the blastocyst stage was assessed at 7-10 days after in vitro fertilization, and the total cells in the blastocysts were counted by staining nuclei with fluorochrome. Some commercial calf sera (CS) and a superovulated cow serum had different effects on the embryonic development to the blastocyst stage (8.6-21.4%), dependent upon their product lots, although the development might not be affected at least by serum progesterone levels. ${\beta}$-Mercaptoethanol (${\beta}$-ME) supplemented into mSOFM was effective to the embryonic development (27.8%), as well as the co-culture system with cumulus cells (19.5%). In a serum- and feeder cell-free culture using mSOFM containing several growth factors and ${\beta}$-ME instead of CS plus co-cultured cumulus cells, bovine serum albumin (BSA, fraction V), but not polyvinyl alcohol (PVA), was highly effective in embryonic development to the blastocyst stage, almost comparable to CS in the serum-contained culture (CS, BSA and PVA; 27.8, 19.5 and 5.7%, respectively). However, fatty acid free BSA rather reduced the number of developed blastocysts, compared with fraction V BSA (7.3 vs 29.4%). In the serum- and feeder cell-free culture, supplement of glucose to the medium (final 2.0 mM) stimulated the cell proliferation of developing embryos 120 hr after in vitro fertilization. These results indicated that a serum-free medium supplemented with ${\beta}$-ME could successfully support the development of bovine one-cell embryos to the blastocyst stage. Moreover, supplement of glucose and fatty acids to the medium might support preferably the development and cell proliferation of embryos.