• Molecules and Cells
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• 제43권7호
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• pp.591-599
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• 2020

Complex cell-to-cell communication underlies the basic processes essential for homeostasis in the given tissue architecture. Obtaining quantitative gene-expression of cells in their native context has significantly advanced through single-cell RNA sequencing technologies along with mechanical and enzymatic tissue manipulation. This approach, however, is largely reliant on the physical dissociation of individual cells from the tissue, thus, resulting in a library with unaccounted positional information. To overcome this, positional information can be obtained by integrating imaging and positional barcoding. Collectively, spatial transcriptomics strategies provide tissue architecture-dependent as well as position-dependent cellular functions. This review discusses the current technologies for spatial transcriptomics ranging from the methods combining mechanical dissociation and single-cell RNA sequencing to computational spatial re-mapping.

• Journal of Yeungnam Medical Science
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• 제4권1호
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• pp.17-23
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• 1987

백서심장조직을 trypsin과 collagenase 두가지의 효소를 사용하여 분리하면서 각 효소의 분리효과와 두가지 효소의 복합적 세포분리효과를 조사하였으며 이와 동시에 dimethyl sulfoxide가 세포분리과정 및 단기간 배양중에 세포에 미치는 효과를 조사한 결과를 다음과 같이 요약할 수 있다. 1. 백서심장조직에서 세포를 분리할 때 $4^{\circ}C$ trypsin 18시간 전처치후 $37^{\circ}C$ collagenase 처치한 군에서 세포생존율 세포수확량 및 심근세포 생존율이 가장 높았다. 즉 단일 효소 처리보다는 효소 복합연속 처리가 더 효율적이었다. 2. $37^{\circ}C$ trypsin만으로 세포분리를 하였을 때 세포생존율과 수확량이 가장 낮았다. 3. 백서심장세포의 분리과정과 초기 배양 과정에 세포회복이나 보호에 대한 DMSO의 효과는 인정되지 않았으며 오히려 세포파괴 효과가 높음을 알 수 있었다.

• 한국발생생물학회지:발생과생식
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• 제16권4호
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• pp.353-361
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• 2012

Human embryonic stem (ES) cells are a potential source of cells for developmental studies and for a variety of applications in transplantation therapies and drug discovery. However, human ES cells are difficult to culture and maintain at a large scale, which is one of the most serious obstacles in human ES cell research. Culture of human ES cells on MEF cells after disassociation with accutase has previously been demonstrated by other research groups. Here, we confirmed that human ES cells (H9) can maintain stem cell properties when the cells are passaged as single cells under a feeder-free culture condition. Accutase-dissociated human ES cells showed normal karyotype, stem cell marker expression, and morphology. We prepared frozen stocks during the culture period, thawed two of the human ES cell stocks, and analyzed the cells after culture with the same method. Although the cells revealed normal expression of stem cell marker genes, they had abnormal karyotypes. Therefore, we suggest that accutase-dissociated single cells can be usefully expanded in a feeder-free condition but chromosomal modification should be considered in the culture after freeze-thawing.

• BMB Reports
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• 제55권3호
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• pp.113-124
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• 2022

Single-cell RNA sequencing (scRNA-seq) has greatly advanced our understanding of cellular heterogeneity by profiling individual cell transcriptomes. However, cell dissociation from the tissue structure causes a loss of spatial information, which hinders the identification of intercellular communication networks and global transcriptional patterns present in the tissue architecture. To overcome this limitation, novel transcriptomic platforms that preserve spatial information have been actively developed. Significant achievements in imaging technologies have enabled in situ targeted transcriptomic profiling in single cells at single-molecule resolution. In addition, technologies based on mRNA capture followed by sequencing have made possible profiling of the genome-wide transcriptome at the 55-100 ㎛ resolution. Unfortunately, neither imaging-based technology nor capture-based method elucidates a complete picture of the spatial transcriptome in a tissue. Therefore, addressing specific biological questions requires balancing experimental throughput and spatial resolution, mandating the efforts to develop computational algorithms that are pivotal to circumvent technology-specific limitations. In this review, we focus on the current state-of-the-art spatially resolved transcriptomic technologies, describe their applications in a variety of biological domains, and explore recent discoveries demonstrating their enormous potential in biomedical research. We further highlight novel integrative computational methodologies with other data modalities that provide a framework to derive biological insight into heterogeneous and complex tissue organization.

• Clinical and Experimental Pediatrics
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• 제48권5호
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• pp.545-550
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• 2005

목 적: 저산소성 허혈성 손상을 받은 신생아의 뇌조직에서 유세포 방법을 통해 세포의 사멸을 분석하기 위해서는 단일세포의 분리가 이루어져야 한다. 본 연구는 세포 분리에 있어서 세포막의 소실을 최소화하고 항원성을 유지하기 위하여 물리적인 방법과 효소 처리를 통한 세포 분리방법의 효율성에 대해 알아보고자 하였다. 방 법 : 생후 7일된 10마리의 SD 신생백서에 우측 경동맥 결찰 후, 8% 산소에 노출시켜 저산소성 허혈증의 손상을 유발하였으며 48시간이 지난 후 뇌조직을 얻어 같은 수의 정상 대조군과 비교하였다. 세포 분리는 물리적인 방법(pipette)과 효소 처리(trypsin 및 collagenase) 방법을 통하여 이루어 졌으며, 세포막의 손상 정도와 범위에 대해서는 annexin V 및 propidium iodide의 형광 염색을 통한 유세포 분석방법을 이용하였다. 결 과 : 정상 대조군에서, 물리적인 방법을 통한 세포 분리가 양반구 모두에서 효소 처리를 한 경우에 비해서 세포의 사멸과 괴사가 통계적으로 유의하게 증가하였다. 저산소성 허혈증을 유발한 군 중, collagenase를 이용하여 세포 분리를 시행한 경우에서 우측 반구의 세포 사멸과 괴사의 비율이 좌측 반구 및 정상 대조군 보다 유의하게 증가하였다. 효소 처리를 통한 세포 분리에서는 서로 유사한 경향을 보였으나, trypsin을 이용한 경우가 collagenase를 이용한 경우에 비해 세포 변화의 정도가 유의하게 감소하였다. 결 론 : 신생아의 뇌조직에서 collagenase를 이용한 단일 세포 분리방법이 세포막의 손상을 최소화하면서 세포막의 성상을 보존할 수 있는 가장 유용한 방법이었다.

• Journal of Electrochemical Science and Technology
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• 제9권3호
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• pp.195-201
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• 2018

To enhance the performance of cathodes at low temperatures, a Cu-coated cathode is prepared, and its electrochemical performance is examined by testing its use in a single cell. At $620^{\circ}C$ and a current density of $150mAcm^{-2}$, a single cell containing the Cu-coated cathode has a significantly higher voltage (0.87 V) during the initial operation than does that with an uncoated cathode (0.79 V). According to EIS analysis, the high voltage of the cell with the Cu-coated cathode is due to the dramatic decrease in the high-frequency resistance related to electrochemical reactions. From XPS analysis, it is confirmed that the Cu is initially in the form of $Cu_2O$ and is converted into CuO after 150 h of operation, without any change in the state of the Ni or Li. Therefore, the high initial cell voltage is confirmed to be due to $Cu_2O$. Because $Cu_2O$ is catalytically active toward $O_2$ adsorption and dissociation, $Cu_2O$ on a NiO cathode enhances cell performance and reduces cathode polarization. However, the cell with the Cu-coated cathode does not maintain its high voltage because $Cu_2O$ is oxidized to CuO, which demonstrates similar catalytic activity toward $O_2$ as NiO.

• Toxicological Research
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• 제20권3호
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• pp.225-232
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• 2004

The suitability of the single cell gel electrophoresis (SCGE) assay as a test for the monitoring of genotoxicity of aquatic environment was evaluated. The SCGE assay was employed to detect DNA damage induced in clam (Spisula sachalinensis) exposed to a direct mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or an indirect mutagen, benzo[a]pyrene (B[a]P). The cells of gill and digestive glands were isolated from clam by homogenization, which was the optimized cell dissociation method, and the level of DNA damage was assessed and expressed as mean tail length. In the gill cells, significant dose- and time-dependent increase was observed in the mean tail length at the concentration from 0.01 to 0.5 ppm MNNG for 96 h. The linear correlation between relative dam-age index (RDI) values was suggested to provide criteria of genotoxicity monitoring for direct acting mutagen. The dose- and time-dependent responses of the digestive glands cells were less sensitive than those of the gill cells. In contrast, the genotoxic response resulting from the exposure of 0.01～1.0 ppm B[a]P to clam revealed a higher sensitivity in the digestive glands cells than the gill cells. The comparison between the time profiles of genotoxic responses in clam and carp, the latter had been obtained in our previous study, indicated that the metabolism of genotoxic compounds in the two aquatic organisms were quite different each other. We conclude that the SCGE assay has the potential as a screening test for routine genotoxicity monitoring of aquatic organisms because of its higher sensitivity and simplicity.

• 미생물학회지
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• 제33권2호
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• pp.131-135
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• 1997

식물조직을 분해하여 단세포화하는 효소, protopectinase를 생산하는 다수의 미생물을 토양에서 분리하였다. 이들 분리된 미생물의 배양여액에서 얻어진 조효소를 감자조직에 처리하여 단세포화된 감자세포를 확인할 수 있었다. 이들 미생물 중에서 비교적 높은 protopectinase의 효소활성을 보이는 균주를 선택하여 형태관찰 결과로부터 Rhizopus sp.임을 알 수 있었다. 선별된 균주 Rhizopus sp. R2의 배양여액에서 얻어진 protopectinase를 이용하여 감자조직의 단세포화를 시도하여 $40^{\circ}C$, pH 4 하에서 최적의 효소반응 조건을 보였다.

• Clinical and Experimental Reproductive Medicine
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• 제37권1호
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• pp.13-23
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• 2010

목 적: 본 연구는 인간 배아줄기세포를 생식세포로의 분화를 유도하고 효소에 의해 분리된 단일 배아줄기세포의 배양조건을 확립하기 위해 수행하였다. 연구방법: Embryonic body (EB)는 배아줄기세포 (hESCs) colony를 떼어내어 3일간 hanging drop culture 방법으로 작성하였고, 이러한 EB를 retionic acid (RA)와 bone morphogenetic protein-4 (BMP4)를 단독 또는 함께 배양액에 첨가하여 14일간 배양함으로써 생식세포로의 분화를 유도하였다. 분화를 유도한 EB는 생식세포 발현유전자인 c-kit과 VASA의 표지인자를 이용한 면역조직형광법으로 분화여부를 조사하였다. 줄기세포는 Collagenase, Tryple 그리고 Accutase 등의 효소로 각각 분리하였고 분리된 단일세포들의 colony formation rate를 조사하였다. 한편 Rho-associated kinase inhibitor (Y-27632)를 단일세포 배양액에 첨가하여 단일세포 분리과정 중에 발생하는 apoptotic damage를 감소시키고자 하였다. 결 과: Tryple 또는 Accutase를 이용하여 분리한 단일세포가 Collagenase에 의해 분리된 세포에 비해 높은 colony formation rate를 나타내었다. 단일세포를 $5{\times}10^3$ cells/well (4 well dish) 농도로 지지세포 위에 seeding하였을 때 다른 농도의 세포를 seeding한 것에 비해 높은 colony formation rate를 확보하는데 효과적이었다. Y27632의 첨가는 단일세포의 colony formation rate를 유의하게 향상시켰으며 특히 Tryple로 분리한 단일세포에 보다 효과적이었다. EB의 분화유도후 c-kit과 VASA의 표지인자를 이용한 면역조직형광염색은 대조군인 정소조직에 비해 약한 형광염색을 나타내었다. 결 론: Tryple을 이용한 단일세포 분리가 건강한 단일세포를 얻는데 가장 효율적이었으며 Y27632 의 첨가는 단일 세포의 생존 및 colony formation에 유익하다는 것을 확인하였다. 다른 연구와는 달리 본 연구에서는 단지 생식세포 표지인자의 희미한 형광염색만을 관찰하였는데 이러한 결과는 본 연구의 분화유도기간이 상대적으로 짧았던 것이 원인이었을 것으로 생각된다.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1996년도 춘계학술대회
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• pp.175-175
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• 1996

The spinal dorsal horn is the area where primary afferent fibers terminate and cutaneous sensory information is Processed. A number of putative neurotransmitter substances, including excitatory and inhibitory amino acids and peptides, are present in this region and sites and cellular mechanisms of their actions have been a target of numerous studies. In this study, single neurons were acutely isolated and the properties of whole cell current and responses to excitatory and inhibitory neurotransmitters were studied by the patch clamp method. Young rats (7-14 days) were anesthetized with diethyl-ether, and the lumbar spinal cord was excised and cut transversely at a thickness of 30$\mu\textrm{m}$ by Vibroslicer. The treatment of spinal slices with low concentration of proteases (pronase and thermolysin 0.75 mg/$m\ell$) and mechanical dissociation yielded isolated neurons with near intact morphology. Multipolar, ellipsoidal and bipolar, and pyramidal cells were shown. By applying step voltage pulses to neurons held at -70 mV, two types of inward currents and one outward currents observed. The fast activating and inactivating inward current was the Na$\^$+/ current because of its fast kinetics and blocking by 0.5${\mu}$M TTX, a specific blocker of Na$\^$+/ channel. The second type of inward currents were sustained. Based on their kinetics and current-voltage relations, it was likely that the second type of inward current was the voltage-dependent Ca$\^$2+/ current. In the presence of TTX, the steady-state currents mainly represented outward K$\^$+/ current which looked like the delayed rectifier K$\^$+/ current. In addition, the membrane currents produced by agonist of excitatory amino acid (EAA) receptor and the endogenous transmitter candidate L-glutamate were recorded in isolated whole-cell voltage clamped neurons as well as responses to inhibitory amino acids (${\gamma}$-amino butyric acid, glycine). Drugs were applied by a method that allows complete exchange of the solution within 1 sec; an infinite number of solutions can be applied to a single cell.