Noninvasive Quality Estimation of Adherent Mammalian Cells for Transplantation

Mutsumi Takagi 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.1

The noninvasive quality estimation of adherent mammalian cells for transplantation is reviewed. The quality and heterogeneity of cells should be estimated before transplantation because cultured cells are not homogeneous but heterogeneous. The estimation of cell quality should be performed noninvasively because most protocols of regenerative medicine are autologous cell system. The differentiation level and contamination of other cell lineage could be estimated by two-dimensional cell morphology analysis and tracking using a conventional phase contrast microscope. The noninvasive determination of the laser phase shift of a cell using a phase-shifting laser microscope, which might be more noninvasive, and more useful than the atomic force microscope and digital holographic microscope, was carried out to determine the three-dimensional cell morphology, and the estimation of the cell cycle phase of each adhesive cell and the mean proliferation activity of a cell population. Chemical analysis of the culture supernatant by conventional analytical methods such as ELISA was also useful to estimate the differentiation level of a cell population. Chemical analysis of cell membrane and intracellular components using a probe beam, an infrared beam, and Raman spectroscopy was useful for diagnosing the viability, apoptosis, and differentiation of each adhesive cell.

Bioelectronics-on-a-chip for cardio myoblast proliferation enhancement using electric field stimulation

Ángel Aragón,María Cebro-Márquez,Eliseo Perez,Antonio Pazos,Ricardo Lage,José Ramón González-Juanatey,Isabel Moscoso,Carmen Bao-Varela,Daniel Nieto 한국생체재료학회 2020 생체재료학회지 Vol.24 No.3

Background: Cardio myoblast generation from conventional approaches is laborious and time-consuming. We present a bioelectronics on-a-chip for stimulating cells cardio myoblast proliferation during culture. Method: The bioelectronics chip fabrication methodology involves two different process. In the first step, an aluminum layer of 200 nm is deposited over a soda-lime glass substrate using physical vapor deposition and selectively removed using a Q-switched Nd:YVO4 laser to create the electric tracks. To perform the experiments, we developed a biochip composed of a cell culture chamber fabricated with polydimethylsiloxane (PDMS) with a glass coverslip or a cell culture dish placed over the electric circuit tracks. By using such a glass cover slip or cell culture dish we avoid any toxic reactions caused by electrodes in the culture or may be degraded by electrochemical reactions with the cell medium, which is crucial to determine the effective cell-device coupling. Results: The chip was used to study the effect of electric field stimulation of Rat ventricular cardiomyoblasts cells (H9c2). Results shows a remarkable increase in the number of H9c2 cells for the stimulated samples, where after 72 h the cell density double the cell density of control samples. Conclusions: Cell proliferation of Rat ventricular cardiomyoblasts cells (H9c2) using the bioelectronics-on-a-chip was enhanced upon the electrical stimulation. The dependence on the geometrical characteristics of the electric circuit on the peak value and homogeneity of the electric field generated are analyzed and proper parameters to ensure a homogeneous electric field at the cell culture chamber are obtained. It can also be observed a high dependence of the electric field on the geometry of the electrostimulator circuit tracks and envisage the potential applications on electrophysiology studies, monitoring and modulate cellular behavior through the application of electric fields.

Effects of tacrolimus on morphology, proliferation and differentiation of mesenchymal stem cells derived from gingva tissue

( Dong Ho Ha ),( Chul Soon Yong ),( Jong Oh Kim ),( Jee Heon Jeong ),( Jun Beom Park ) 영남대학교 약품개발연구소 2016 영남대학교 약품개발연구소 연구업적집 Vol.26 No.-

Tacrolirnus is a 23-membered macrolide lactone with potent immunosuppressive activity that is effective in the prophylaxis of organ rejection following kidney, heart and liver transplantation. Tacrolimus also exerts a variety of actions on bone metabolism. The aim of the present study was to evaluate the effects of different concentrations of tacrolimus on the morphology and viability of human stem cells derived from the gingiva. Gingival-derived stem cells were grown in the prcsence of tacrolimus at final concentrations ranging from 0.001 to 100 μg/ml. The morphology of the cells was viewed under an inverted microscope and the cell viability was analyzed using Cell Counting kit-S (CCK-8) on days 1,3,5 and 7. Alizarin Red S staining was used to assess mineralization of treated cells. The control group showed spindle-shaped, fibroblast-like morphology and the shapes of the cells in 0.001, 0.01,0.1,1 and 10 μg/ml tacrolimus were similar to those of the control group. All groups except the 100 μg/ml group showed increased cell proliferation over time. Cultures grown in the presence of tacrolimus at 0.001, 0.01, 0.1, I and 10 μg/ml were not identified to be significantly different compared with the control at days 1, 3 and 5 using the CCK-8 assays. Increased mineralized deposits were noted with increased incubation time. Treatment with tacrolimus from 0.001 to 1 μg/mlled to an increase in mineralization compared with the control group. Within the limits of this study, tacrolimus at the tested concentrations (ranging from 0.001 to 10 μg/ml) did not result in differences in the viability of stem cells derived from gingiva; however it did enhance osteogenic differentiation of the stem cells.

CD29 및 CD98 활성 매개에 의한 Jurkat T 세포의 유착과 그 활용

김병훈,조재열,Kim, Byung-Hun,Cho, Jae-Youl 대한약학회 2009 약학회지 Vol.53 No.3

Cell-cell adhesion managed by various adhesion molecules plays an important role in regulating functional activation of cells. This event mediates attachment of inflammatory cells to endothelial cells, interaction of antigen-presenting cells with T cells and metastatic adherence of cancer cells to epithelial tissue cells. Therefore, this cellular response is considered as one of therapeutic target to treat various cancers and inflammatory diseases. To develop proper model for evaluation of functional activation of adhesion molecules, the ability of U937 and Jurkat T cells responsive to various adhesion inducers such as phorbal-12-myristate-13-acetate (PMA), staurosporin and monoclonal antibodies to CD29, CD43 and CD98 was investigated using quantitative cell-cell adhesion assay. U937 cells made more cell-cell clusters by the treatment of antibodies to CD29 and CD43 than Jurkat T cells, while Jurkat T cells exhibited increased cell-cell adhesion ability in CD98 antibody treatment. In agreement, the surface levels of CD29 and CD98 were highly observed in U937 and Jurkat T cells, respectively. Therefore, our data suggest that Jurkat T and U937 cells can be used for model system to evaluate functional activation of adhesion molecules such as CD29 and CD98.

Facile endothelial cell micropatterning induced by reactive oxygen species on polydimethylsiloxane substrates

Choi, Jong Seob,Kim, Do Hyun,Seo, Tae Seok Elsevier 2016 Biomaterials Vol.84 No.-

<P><B>Abstract</B></P> <P>Sophisticated cell pattern provides unique cellular assay platform for studying cell to cell interaction, cellular differentiation and signaling, high-throughput cell response to chemicals. In this study, we demonstrated reactive oxygen species (ROS) mediated endothelial cell micropatterning on a polydimethylsiloxane (PDMS) substrate. The exposure of UV/O radiation led to the formation of ROS on the surface of PDMS, which could selectively prevent adhesion of endothelial cells. The degree of ROS amount was monitored according to the UV/O irradiation time, and at least 36 μM of ROS resulted in the precise cellular micropattern on the PDMS. The presence of ROS affected not only cellular detachment from the substrate, but also endothelial cell morphology such as cell spreading area, confluence, nuclear area and nuclear inverse aspect ratio. In addition, we could observe that the actin cytoskeleton of cells was also constricted due to ROS, thereby minimizing the focal adhesion area of vinculin. Compared with previously reported methods which use chemical treatment or nano/microstructure on the substrate, the proposed methodology is quite simple, accurate, and harmless to the patterned endothelial cells.</P>

Coculture Effects on the Osteogenic Differentiation of Human Mesenchymal Stromal Cells

Astghik Hayrapetyan,Soraya Surjandi,Evita EPJ Lemsom,Marleen MMW Wolters,John A Jansen,Jeroen JJP van den Beucken 한국조직공학과 재생의학회 2016 조직공학과 재생의학 Vol.13 No.6

Cell-based bone regeneration is generally pursued based on single cell type approaches, for which human adipose tissue-derived mesenchymal stromal cells (AT-MSCs) are frequently used, owing to their easy accessibility and relatively large yield. In view of multiple cell types involved in physiological bone regeneration, this study aimed to evaluate the osteogenic differentiation of AT-MSCs upon co-culture with endothelial cells or macrophages in a direct or indirect in vitro co-culture set-up. Our hypotheses were that 1) endothelial cells and macrophages stimulate AT-MSCs proliferation and osteogenic differentiation and that 2) these two cell types will more profoundly affect osteogenic differentiation of AT-MSCs in a direct compared to an indirect co-culture set-up, because of the possibility for both cell-cell interactions and effects of secreted soluble factors in the former. Osteogenic differentiation of AT-MSCs was stimulated by endothelial cells, particularly in direct co-cultures. Although initial numbers of AT-MSCs in co-culture with endothelial cells were 50% compared to monoculture controls, equal levels of mineralization were achieved. Macrophages showed a variable effect on AT-MSCs behavior for indirect co-cultures and a negative effect on osteogenic differentiation of AT-MSCs in direct co-cultures, the latter likely due to species differences of the cell types used. The results of this study demonstrate potential for cell combination strategies in bone regenerative therapies.

인체거대세포바이러스에 의한 인체 단핵구세포의 세포주기 저해

장소영,김미숙,이찬희,Jang, So-Young,Kim, Mi-Suk,Lee, Chan-Hee 한국미생물학회 2008 미생물학회지 Vol.44 No.4

마이엘로이드 계열의 단핵세포는 인체세포거대바이러스(human cytomegalovirus, HCMV)의 잠복 부위로 알려져 있다. 다양한 세포에서 HCMV에 의한 세포주기의 촉진 또는 억제에 관한 연구는 많이 있었지만, 단핵세포에서의 연구는 거의 없는 상태이다. 이에 본 연구에서는 단핵세포에 HCMV가 감염되면 세포주기에 어떤 변화가 나타나는지 알아보고자 하였다. Propidium iodide를 이용한 유세포 분석을 통한 세포주기 분석에서 HCMV에 감염된 THP-1 세포에서는 G0-G1기가 증가하고, 그만큼 S가 감소함을 보았다. 반면 HL-60 세포에서는G0-G1기와 S기의 상대적인 비율에 큰 변화가 없었다. BrdU 유입 실험에서 THP-1세포의 DNA 합성이 HCMV 감염에 의해 억제됨을 알 수 있었다. 세포주기의 G1기에서 S기로의 전환을 억제하는 p21 단백질은HCMV에 감염된 THP-1 세포에서는 발현이 유도되었지만 HL-60 세포에서는 거의 발현이 되지 않았다. 따라서 HCMV는 promocyte THP-1 세포에서 p21 단백질의 유도에 의해 세포주기를 G0-G1기에서 억류함에 따라 세포중식을 억제하는 것으로 생각된다. Monocytic cells in myeloid lineage are known for latent site of HCMV Previous studies have suggested that HCMV regulates cell cycle progression in a variety of cells, but studies in monocytic cells are limited. In this study, we attempted to understand cell cycle changes after HCMV infection in the monocytic cell lines. Flow cytometric analyses using propidium iodide revealed that the proportion of G0-G1 phase was increased and the proportion of S phase decreased in HCMV-infected THP-1 cells, but not in HL-60 cells. BrdU-incorporation assay supported that cell proliferation was inhibited in HCMV-infected THP-1 cells by inhibition of de novo DNA synthesis. Western blot analysis revealed that p21, inhibitor of cell cycle progression from G1 phase to S phase, was induced in HCMV-infected THP-1 cells but not in HL-60 cells. Thus, HCMV inhibited cell pro-liferation by arresting the cell cycle at G0-G1 phase through induction of p21 protein in promocytic THP-1 cells.

A Lignin Molecular Brace Controls Precision Processing of Cell Walls Critical for Surface Integrity in <i>Arabidopsis</i>

Lee, Yuree,Yoon, Taek Han,Lee, Jiyoun,Jeon, So Yeon,Lee, Jae Ho,Lee, Mi Kyoung,Chen, Huize,Yun, Ju,Oh, Se Yun,Wen, Xiaohong,Cho, Hui Kyung,Mang, Hyunggon,Kwak, June M. Elsevier 2018 Cell Vol.173 No.6

<P><B>Summary</B></P> <P>The cell wall, a defining feature of plants, provides a rigid structure critical for bonding cells together. To overcome this physical constraint, plants must process cell wall linkages during growth and development. However, little is known about the mechanism guiding cell-cell detachment and cell wall remodeling. Here, we identify two neighboring cell types in <I>Arabidopsis</I> that coordinate their activities to control cell wall processing, thereby ensuring precise abscission to discard organs. One cell type produces a honeycomb structure of lignin, which acts as a mechanical “brace” to localize cell wall breakdown and spatially limit abscising cells. The second cell type undergoes transdifferentiation into epidermal cells, forming protective cuticle, demonstrating <I>de novo</I> specification of epidermal cells, previously thought to be restricted to embryogenesis. Loss of the lignin brace leads to inadequate cuticle formation, resulting in surface barrier defects and susceptible to infection. Together, we show how plants precisely accomplish abscission.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Two neighboring cell types coordinate cellular activities for organ separation </LI> <LI> A honeycomb structure of lignin acts as a “molecular brace” </LI> <LI> The lignin brace spatially restricts cell wall breakdown </LI> <LI> Lignin deposition ensures surface integrity of transdifferentiated epidermal cells </LI> </UL> </P> <P><B>Graphical Abstract</B></P> <P>[DISPLAY OMISSION]</P>

Monitoring of cell-cell interaction in a programmable static droplet array with a confined microenvironment

김동영,진시형,이병진,정성근,이성식,( Matthias Peter ),이창수 한국공업화학회 2019 한국공업화학회 연구논문 초록집 Vol.2019 No.0

Cell-cell interaction can occur through biochemical signaling molecules, regulating a variety of biological responses such as quorum sensing, biofilm formation, and inflammatory response. However, traditional cell culture systems are difficult to accurately reproduce the effects of cell-cell interactions and mixed cell populations, have no single cell resolution, and often have limited sensitivity and accuracy. Thus, there is a growing need for a novel platform for isolating, manipulating, tracking, and imaging cells to better study the various cell-cell interaction scenarios. Here we present a programmable static droplet array (SDA) for monitoring of the effect of population on natural cell-cell contact and signaling processes in a confined microenvironment. The SDA provides a valuable research tool to study cell-cell communication and signaling in a controlled microenvironment with the sensitivity and accuracy required for screening and long-term phenotypic analysis.

Effects of nicotine on proliferation, cell cycle, and differentiation in immortalized and malignant oral keratinocytes

Lee, Hwa-Jeong,Guo, Han-Young,Lee, Sun-Kyung,Jeon, Byung-Hun,Jun, Chang-Duk,Lee, Suk-Keun,Park, Myung-Hee,Kim, Eun-Cheol Munksgaard International Publishers 2005 Journal of oral pathology & medicine Vol.34 No.7

<P>Background: </P><P>Numerous epidemiological studies have reported that tobacco smoking is a major risk factor for oral cancer, but relatively little is known about the effect of nicotine, a major product of cigarette smoking, on immortalized oral keratinocytes and cancer cells.</P><P>Methods: </P><P>We investigated the effects of nicotine on the growth and differentiation of immortalized human oral keratinocytes (IHOK), primary oral cancer cells (HN4), metastatic oral cancer cells (HN12), and human skin keratinocytes (HaCaT), in the monolayer and in the three-dimensional (3D) raft cultures using the MTT assay, Western blotting, and cell cycle analysis.</P><P>Results: </P><P>Nicotine inhibited the proliferation of immortalized and malignant keratinocytes in dose- and time-dependent manners as determined by MTT assay. The 3D organotypic culture showed that nicotine at high concentration (300 <I>μ</I>M) inhibits epithelial maturation, surface keratinization, and decreased epithelial thickness. Flow cytometry showed that nicotine inhibited cell cycle progression by inducing G<SUB>0</SUB>/G<SUB>1</SUB> arrest of HaCaT, IHOK, HN4, and HN12 cells without causing apoptosis. Nicotine treatment increased p21 expression in immortalized cells (HaCaT, IHOK) and oral cancer cells (HN4, HN12), but decreased pRb and p53 expression in oral cancer cells. Moreover, after high-dose nicotine treatment, the involucrin expression increased markedly in immortalized cells, but not in oral cancer cells.</P><P>Conclusions: </P><P>We demonstrated that nicotine inhibits growth through cell cycle arrest at G<SUB>0</SUB>/G<SUB>1</SUB> phase probably by increasing the expression of p21<SUP>WAF1/CIP1</SUP>. Nicotine also affects epithelial differentiation in immortalized and malignant oral keratinocytes. Malignant oral keratinocytes appear to be more resistant to the effects of nicotine on epithelial growth and differentiation as compared to the immortalized cells.</P>