Osteogenic Response of MC3T3-E1 and Raw264.7 in the 3D-Encapsulated Co-Culture Environment

Kim Jungju,Lyu Hao-Zhen,Jung Chisung,Lee Kyung Mee,Han Shi Huan,Lee Jae Hyup,Cha Misun 한국조직공학과 재생의학회 2021 조직공학과 재생의학 Vol.18 No.3

Background: Three-dimensional (3D) in vitro cultures recapitulate the physiological microenvironment and exhibit high concordance with in vivo conditions. Improving co-culture models with different kind of cell types cultured on a 3D scaffold can closely mimic the in vivo environment. In this study, we examined the osteogenic response of pre-osteoblast MC3T3-E1 cells and Raw264.7 mouse monocytes in a 3D-encapsulated co-culture environment composed of the Cellrix® 3D culture system, which provides a physiologically relevant environment. Methods: The Cellrix® 3D Bio-Gel scaffolds were used to individually culture or co-culture two type cells in 3D microenvironment. Under 3D culture conditions, osteoblastic behavior was evaluated with an ALP assay and staining. ACP assay and TRAP staining were used as osteoclastic behavior indicator. Results: Treatment with osteoblastic induction factors (+3F) and RANKL had on positively effect on alkaline phosphatase activity but significantly inhibited to acid phosphatase activity during osteoclastic differentiation in 3D co-culture. Interestingly, alkaline phosphatase activity or acid phosphatase activity in 3D co-culture was stimulated with opposite differentiation factors at an early stage of differentiation. We guess that these effects may be related to RANK–RANKL signaling, which is important in osteoblast regulation of osteoclasts. Conclusion: In this study, the osteogenic response of 3D encapsulated pre-osteoblast MC3T3-E1 cells and mouse monocyte Raw264.7 cells was successfully demonstrated. Our 3D culture conditions will be able to provide a foundation for developing a high-throughput in vitro bone model to study the effects of various drugs and other agents on molecular pathways. Background: Three-dimensional (3D) in vitro cultures recapitulate the physiological microenvironment and exhibit high concordance with in vivo conditions. Improving co-culture models with different kind of cell types cultured on a 3D scaffold can closely mimic the in vivo environment. In this study, we examined the osteogenic response of pre-osteoblast MC3T3-E1 cells and Raw264.7 mouse monocytes in a 3D-encapsulated co-culture environment composed of the Cellrix® 3D culture system, which provides a physiologically relevant environment. Methods: The Cellrix® 3D Bio-Gel scaffolds were used to individually culture or co-culture two type cells in 3D microenvironment. Under 3D culture conditions, osteoblastic behavior was evaluated with an ALP assay and staining. ACP assay and TRAP staining were used as osteoclastic behavior indicator. Results: Treatment with osteoblastic induction factors (+3F) and RANKL had on positively effect on alkaline phosphatase activity but significantly inhibited to acid phosphatase activity during osteoclastic differentiation in 3D co-culture. Interestingly, alkaline phosphatase activity or acid phosphatase activity in 3D co-culture was stimulated with opposite differentiation factors at an early stage of differentiation. We guess that these effects may be related to RANK–RANKL signaling, which is important in osteoblast regulation of osteoclasts. Conclusion: In this study, the osteogenic response of 3D encapsulated pre-osteoblast MC3T3-E1 cells and mouse monocyte Raw264.7 cells was successfully demonstrated. Our 3D culture conditions will be able to provide a foundation for developing a high-throughput in vitro bone model to study the effects of various drugs and other agents on molecular pathways.

성장생물학 : 지방과 근육 세포주의 단독 및 공동배양을 통한 세포형태학 및 세포물질 비교 연구

연성흠 ( Seong Heum Yeon ),조원모 ( Won Mo Cho ),백경훈 ( Kyung Hoon Baek ),송만강 ( Man Kang Song ),최창원 ( Chang Weon Choi ),황보순 ( Bo Soon Hwang ),박성권 ( Sung Kwon Park ) 한국동물자원과학회 ( 구 한국축산학회 ) 2012 한국축산학회지 Vol.54 No.2

본 연구는 기존 단독배양 위주로 이루어져온 세포배양 연구의 방법학적 한계의 극복과 대안을 제시하고자 지방과 근육세포주의 단독 및 공동배양에서 배양기법에 따른 지방 및 근육세포의 분화에 미치는 영향을 비교 조사하고자 실시하였다. 3T3-L1(지방세포) 및 L6(근육세포) 세포주는 성장배지인 10% FBS/DMEM(1% Pen- Strep solution 및 0.1% Fungizone 첨가) 하에서 48h 동안 단독배양 후 5% FBS/DMEM에서 배양하였다. 분화를 위한 단독 및 공동배양에서는 지방 및 근육세포 모두 분화유도물질 없이 2% FBS/DMEM으로 배양하였고, 공동배양에서는 0.4㎛ insert membrane을 사용하여 6 well plate 하단에 L6 cell을, 상단에는 3T3-L1 cell을 공생시켰다. 지방 및 근육세포 분화정도 측정은 세포별 형태학적 측정과 glycerol-3-phosphate dehydrogenase (GPDH) 및 creatine kinase(CK) 분석을 통해 조사되었다. 형태학적으로 볼때 3T3-L1 세포주는 공동배양보다 단독배양 시 분화가 더욱 잘 일어났고 L6 세포주의 경우 역으로 같았다. 세포물질분석에서는 분화배지 처리일(day 0)과 비교해 단독 및 공동배양 모두 지방세포 내 GPDH의 활성도가 유의적으로(P<0.05) 증가했음을 확인할 수 있었고 단독배양이 공동배양보다 유의적으로(P<0.05) 높은 수준의 GPDH 활성도를 보였다. L6 역시 마찬가지로 분화배지 처리일에 비하여 단독 및 공동배양 모두 CK 활성도가 유의적으로(P<0.05) 높았고, CK 활성도가 공동배양에서 유의적으로(P<0.05) 높게 나타남을 확인할 수 있었다. 이러한 결과는 기존 연구에서 이용된 단독 배양을 통한 세포 분화 결과 등은 생체와 비교 시 방법학적 한계로 인해 실제 생체 내에서는 그 분화정도가 매우 다를 것으로 생각되며, 이것은 앞으로 정확한 세포배양 결과 확보를 위해서는 단독배양보다는 공동배양기법을 사용해야 함을 의미한다. 향후 다양한 조건과 분화조절 물질들의 첨가를 통한 추가적인 공동배양실험이나 지방분화관련 분자생물학적 물질분석 등 다양한 실험 수행 시 보다 현실적이고 대량의 기초자료 확보가 가능할 것으로 판단된다. Present study was performed to investigate the effect of single and co-culture of adipocyte and muscle cell lines on cell differentiation. 3T3-L1(adipocyte) and L6(muscle) cell lines were single-cultured on the condition of 10% fetal bovine serum (FBS)/Dulbeco`s modified eagle`s medium(DMEM) for 48 h followed by culture within 5% FBS/DMEM as a growth media. Then, the growth media was replaced by differentiation media composed of 2% FBS/DMEM without additives in single- or co-culture of the 3T3-L1 and the L6 cells to induce differentiation of both cell types. In co-culture system, the 3T3-L1 and the L6 cells were grown in separated places by being seeded on a 0.4μm insert membrane and on the bottom of 6 well plate, respectively. Cell differentiation was measured using morphological investigation and cytosolic analysis of glycerol-3-phosphate dehydrogenase(GPDH; for 3T3-L1) and creatine kinase(CK; for L6). Based on the GPDH results, the presence of L6 cells did not stimulate 3T3-L1 differentiation showing more differentiation of 3T3-L1 cells in the single-culture compared to the co-culture condition. In contrast, 3T3-L1 cells in the co-culture promoted differentiation of L6 cells. Enzymatic analysis supported this result showing that 3T3-L1 cells showed statistically(P<0.05) higher GPDH activity in the single-culture than the co-culture, whereas CK results of L6 cells were vice versa(P<0.05). Overall, present results may indicate that co-culture system is more reliable and precise technique compared to single-culture. Further studies on several co-culture trials including different media conditions, supplementation of differentiating substances, molecular biological analysis, etc. should be required to obtain practical and fundamental mass data.

Removal of nutrients and COD through co-culturing activated sludge and immobilized <i>Chlorella vulgaris</i>

Mujtaba, Ghulam,Rizwan, Muhammad,Kim, Garam,Lee, Kisay Elsevier 2018 CHEMICAL ENGINEERING JOURNAL -LAUSANNE- Vol.343 No.-

<P><B>Abstract</B></P> <P>Simultaneous removal of inorganic nutrients (nitrogen and phosphorus) and organic carbon (glucose) was realized from synthetically-made municipal wastewater using co-culture of suspended activated sludge and immobilized <I>Chlorella vulgaris</I> in a single reactor. Use of immobilized microalgae and suspended activated sludge can help in separating microalgae biomass from the culture broth at the end of treatment. Efficient removal of nutrients and COD was achieved by the symbiotic co-culture than stand-alone cultures of <I>C. vulgaris</I> and activated sludge after 2-d of retention time. By decreasing inoculum ratio of suspended activated sludge to immobilized <I>Chlorella vulgaris</I> (from 5.0 to 0.2), the performance of nutrients removal was enhanced significantly, and the co-culture at 0.5 inoculum ratio accomplished highest removal of nitrogen (99.8%) and phosphorus (100%) within 2 days of incubation. In the case for the removal of COD, co-culture with different inoculum ratios demonstrated virtually similar performance (showing 90–95% removal after 2-d treatment) which is indicating that there was no robust association between carbon degradation and inoculum ratio. The maximum growth of microalgae (0.76 from initial 0.4 g L<SUP>−1</SUP>) was obtained from the co-culture having 0.5 inoculum ratio indicating significance of proper inoculation. Co-culture at 0.5 inoculum ratio of suspended activated sludge and immobilized <I>C. vulgaris</I> was advanced in the semi-continuous mode treatment. By repeating batch cycles for three times, the co-culture considerably eliminated 98–100% nitrogen, 92–100% phosphorus, and 94–96% COD and accumulated 2.2 g L<SUP>−1</SUP> of biomass production confirming the stability of the current co-culture system in municipal wastewater medium.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The current co-culture exhibited simultaneous removal of nutrients and COD in two days. </LI> <LI> Removal efficiency was increased by decreasing the inoculum ratio from 5.0 to 0.5. </LI> <LI> The co-culture system demonstrated stability up to three repeated batch operations. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Co-culture-based biological carbon monoxide conversion by <i>Citrobacter amalonaticus</i> Y19 and <i>Sporomusa ovata</i> via a reducing-equivalent transfer mediator

Lee, Cho Rong,Kim, Changman,Song, Young Eun,Im, Hyeonsung,Oh, You-Kwan,Park, Sunghoon,Kim, Jung Rae Elsevier 2018 Bioresource technology Vol.259 No.-

<P><B>Abstract</B></P> <P>The biological conversion of carbon monoxide (CO) has been highlighted for the development of a C1 gas biorefinery process. Despite this, the toxicity and low reducing equivalent of CO uptake make biological conversion difficult. The use of synthetic co-cultures is an alternative way of enhancing the performance of CO bioconversion. This study evaluated a synthetic co-culture consisting of <I>Citrobacter amalonaticus</I> Y19 and <I>Sporomusa ovata</I> for acetate production from CO. In this consortium, the CO<SUB>2</SUB> and H<SUB>2</SUB> produced by the water-gas shift reaction of <I>C. amalonaticus</I> Y19, were utilized further by <I>S. ovata</I>. Higher acetate production was achieved in the co-culture system compared to the monoculture counterparts. Furthermore, syntrophic cooperation via various reducing equivalent carriers provided new insights into the synergistic metabolic benefits with a toxic and refractory substrate, such as CO. This study also suggests an appropriate model for examining the syntrophic interaction between microbial species in a mixed community.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Co-culture of <I>C. amalonaticus</I> Y19 and <I>S. ovata</I> was examined using CO as substrate. </LI> <LI> CO was converted to CO<SUB>2</SUB> and H<SUB>2</SUB> by Y19, then metabolized by ovata. </LI> <LI> Co-culture presented better CO consumption and acetate production than mono-culture. </LI> <LI> The addition of HNQ as mediator improves conversion of CO to acetate. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

3차원 Co-culture 시스템을 통한 BMSC의 NP-like Cell로의 분화

김동화,김수향,허수진,신지원,김영직,박소희,전재우,신정욱,Kim, D.H.,Kim, S.H.,Heo, S.J.,Shin, J.W.,Kim, Y.J.,Park, S.H.,Jun, J.W.,Shin, J.W. 대한의용생체공학회 2008 의공학회지 Vol.29 No.2

The goal of this study is to investigate the effect and potential of three-dimensional Co-culture of BMSCs (bone marrow stromal Cells) and NP (nucleus pulposus) Cells on the differentiation of BMSCs into NP-like Cells. The NP Cells and BMSCs were isolated and cultured from New Zealand White rabbits. The isolated NP Cells and BMSCs were prepared in different alginate beads. Those two types of beads were separated by a track-etched membrane of $3\;{\mu}m$ pore in a 6-well culture plate. No growth factors were used. In addition to these, NP and BMSC were cultured in the beads independently for control. The number of Cells in Co-culturing system was half of those in two control groups. Proliferation and production of glycosaminoglycan (GAG) were evaluated along with histological observation. The GAG production rate(GAG contents/Cell) of Co-cultured BMSCs were much higher than that of BMSCs cultured alone. The total amounts of GAG produced by BMSCs in Co-culturing system were larger than those produced by BMSCs in control group and were comparable with those produced by NP alone even the number of each Cell was half of BMSCs in Co-culturing system. This study showed the potential of differentiation of BMSCs into NP-like Cells through three-dimensional Co-culture system even without any chemical agents.

Influence of co-culturing muscle satellite cells with preadipocytes on the differentiation of adipocytes and muscle cells isolated from Korean native cattle

최창원 충남대학교 농업과학연구소 2018 Korean Journal of Agricultural Science Vol.45 No.4

The present study was done to investigate the effect of co-culturing muscle satellite cells (MSCs) and intramuscular preadipocytes (IPs) on the differentiation of adipocytes and muscle cells isolated from Korean native cattle. MSCs and IPs were single-cultured in 10% fetal bovine serum/Dulbecco's modified Eagles medium (FBS/DMEM) for 48 h followed by culturing in 5% FBS/DMEM as the growth media. Then, the growth media was replaced by differentiation media composed of 2% FBS/DMEM without any additives for the single- or co-culture of muscle cells and intramuscular adipocytes to induce the differentiation of both cell types. Cell differentiation was measured by morphological investigation and cytosolic enzyme analysis of glycerol-3-phosphate dehydrogenase (GPDH) for the adipocytes and creatine kinase (CK) for the muscle cells. In the morphological test, the presence of muscle cells did not stimulate adipocyte differentiation showing more differentiation of the adipocytes in the single-culture compared to the co-culture condition. However, the differentiation of muscle cells was promoted by adipocytes in the co-culture. The results of the enzymatic analysis were highly associated with the morphological results with a statistically higher GPDH activity (p < 0.05) appearing in the single-culture than in the co-culture, whereas the opposite was true for the CK activity of the muscle cells (p < 0.05). By manipulating in vivo the milieu using a co-culture, we could detect the difference in the rate of cell differentiation and suggest that a co-culture system is a more reliable and precise technique compared to a single-culture. Further studies on various co-culture trials including supplementation of differentiating substances, gene expression analysis, etc. should be done to obtain practical and fundamental data.

Effects of a Co-culture of Scenedesmus dimorphus and Mixed Bacteria on the Organic Matter and N, P Removal Efficiencies Evaluation in Mixotrophic Conditions

( Hyeon-jeong Hwang ),( Kyoungjin Choi ),( Shan Zhang ),( Eun-ji Kim ),( Jaehyoung Cho ),( Sun-jin Hwang ) 한국폐기물자원순환학회(구 한국폐기물학회) 2015 한국폐기물자원순환학회 춘계학술발표논문집 Vol.2015 No.-

This study aims to increase the organic carbon, nitrogen and phosphorus removal efficiencies than the conventional method and meet higher effluent water quality standards by co-culture with bacteria in activated sludge in the aeration tank. By the co-culture in mixotrophic conditions through metabolic characterization of Scenedesmus dimorphus and bacteria in the aeration activated sludge, assessed how the impact on the organic matter, nitrogen and phosphorus removal rate. In addition, the study was to determine if it is possible to supply the oxygen necessary for the bacteria through the photosynthesis of algae without aeration. To test the synergistic effects of the co-culture, we compared co culture with S. dimorphus - only, Bacteria - only as a controls. In the co-culture condition inoculation ratio is based on the TSS concentration S. dimorphus and bacteria 1 : 5 (w / w), 5 : 5 (w / w) at a ratio. The growth rate of S. dimorphus - only condition was higher, which was 10 times and 2 times lower compared to B : A (5 : 1), B : A (5 : 5) respectively. Organic carbon removal rate of S. dimorphus - only condition was lower than other conditions, the remaining conditions were consumed at a similar rate. In the co-culture of Mixed bacteria and S. dimorphus, ammonium and phosphate removal rate has been high. But, nitrate removal rate showed a tendency to decrease compared to the Bacteria-only condition. When considering the Ammonium, nitrate and phosphate removal rate, N, P removal efficiency which is most excellent in terms of bacteria, such as S. dimorphus ratio (5 : 5) were co-cultured in the conditions. DO was maintained at 3 mg/L or more under all conditions. So, implying co-culture has the potential to decrease aeration costs.

Enhancement of Laccase Production from Wood-Rotting Fungus by Co-Culture with Trichoderma longibrachiatum

( Hyun-Chae Jung ) 한국목재공학회 2019 목재공학 Vol.47 No.2

This work aimed to evaluate the influence of culture conditions on laccase production in the co-culture of wood-rotting fungus with Trichoderma sp. The effects of infection extent, infection time, and culture filtrate of Trichoderma sp. on the laccase production by wood-rotting fungus in co-culture were examined. T. rubrum LKY-7 and T. longibrachiatum were selected as fungi which are effective in co-culture for laccase production. A significant increase in laccase activity was observed when T. rubrum LKY-7 was co-cultured with T. longibrachiatum in glucose-peptone liquid medium, yielding an increase of more than 5 times in laccase activity, as compared with control. Laccase production by T. rubrum LKY-7 during co-culturing was significantly influenced by the infection extent and the infection time of T. longibrachiatum. Maximal laccase activity was obtained when T. rubrum LKY-7 culture was infected by T. longibrachiatum after 3 days of cultivation at an inoculum size ratio of 0.5 to 1. The addition of culture filtrate or autoclaved mycelium of T. longibrachiatum to T. rubrum LKY-7 culture did not significantly enhance laccase production by T. rubrum LKY-7 as compared with control (mono cultures of T. rubrum LKY-7).

신경세포가 별아교세포의 아교섬유성 산단백질 표현에 미치는 영향

배형미,박정선,연동수,Bae Hyung-Mi,Park Jung-Sun,Yeon Dong-Soo 대한약리학회 1997 The Korean Journal of Physiology & Pharmacology Vol.1 No.3

Injury to brain transforms resting astrocytes to their reactive form, the hallmark of which is an increase in glial fibrillary acidic protein (GFAP), the major intermediate filament protein of their cell type. The overall glial response after brain injury is referred to as reactive gliosis. Glial-neuronal interaction is important for neuronal migration, neurite outgrowth and axonal guidance during ontogenic development. Although much attention has been given to glial regulation of neuronal development and regeneration, evidences also suggest a neuronal influence on glial cell differentiation, maturation and function. The aim of the present study was to analyze the effects of glial-hippocampal neuronal co-culture on GFAP expression in the co-cultured astrocytes. The following antibodies were used for double immunostaining chemistry; mouse monoclonal antibodies for confirm neuronal cells, rabbit anti GFAP antibodies for confirm astrocytes. Primary cultured astrocytes showed the typical flat polygonal morphology in culture and expressed strong GFAP and vimentin. Co-cultured hippocampal neurons on astrocytes had phase bright cell body and well branched neurites. About half of co-cultured astrocytes expressed negative or weak GFAP and vimentin. After 2 hour glutamate (0.5 mM) exposure of glial-neuronal co-culture, neuronal cells lost their neurites and most of astrocytes expressed strong CFAE and vimentin. In Western blot analysis, total GFAP and vimentin contents in co-cultured astrocytes were lower than those of primary cultured astrocytes. After glutamate exposure of glial-neuronal co-culture, GFAP and vimentin contents in astrocytes were increased to the level of primary cultured astrocytes. These results suggest that neuronal cell decrease GFAP expression in co-cultured astrocytes and hippocampal neuronal-glial co-culture can be used as a reactive gliosis model in vitro for studying GFAP expression of astrocytes.

협동학습을 활용한 한국어 교재의 문화 텍스트 교육 방안 -자율적 협동학습(Co-op Co-op) 모형을 중심으로-

이영주 한국어교육연구학회 2023 한국어교육연구 Vol.- No.20

본 연구의 목적은 학습자 중심의 협동학습(cooperative learning)을 활용하여 문화교육의 방법론적 관점에서 한국어 문화 수업에 실제 적용할 수 있는 교수·학습법을 구안하는 데 있다. 이를 위해 먼저 협동학습의 개념과 원리를 알아보았으며, 협동학습을 적용한 한국어 교육의 연구와 논의를 살펴 협동학습을 기반으로 하는 한국어 문화 수업의 필요성을 파악하였다. 협동학습의 여러 모형 중 자율적 협동학습(Co-op Co-op)의 원리와 수업 절차, 기대 효과 등을 바탕으로 한국어 문화 수업의 틀을 잡고 문화 수업을 설계하였다. 자율적 협동학습(Co-op Co-op) 모형을 적용한 한국어 문화 수업을 통해, 한국어 학습자가 학습하고 습득한 문화적 지식과 경험이 내재화되고 학업적 성취와 더불어 상호 문화 의사소통 능력까지 함양될 수 있을 것으로 기대한다. 차후 자율적 협동학습(Co-op Co-op)의 모형을 활용한 한국어 문화 실험 수업을 통해 협동학습이 학습자의 학업 성취도, 자기 효능감, 학습 태도 등에 미치는 영향에 관한 연구를 이어갈 예정이다. The purpose of this study is to devise teaching and learning methods that can be applied to Korean culture classes from the methodological perspective of cultural education using learner-centered cooperative learning. To this end, the concept and principles of cooperative learning were first examined, and the research and discussion of Korean language education applying cooperative learning were examined to understand the necessity of Korean language culture classes based on cooperative learning. Among the various models of cooperative learning, based on the principles of Co-op Co-op model, instructional procedures, and expected effects, the Korean language and culture classes were designed. It is expected that the cultural knowledge and experiences learned and acquired by Korean learners will be internalized and academic achievements as well as mutual cultural communication skills will be cultivated through Korean language culture classes applying the Co-op Co-op model. In the future, we will continue research on the impact of cooperative learning on learners' academic achievement, self-efficacy, and learning attitudes through Korean language culture experimental classes using models of Co-op Co-op.