Experimental and Therapeutic Medicine
·
2014
·Vol. 8
·期 3
·935-942
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IF 0.941
Wei Zhang,Jiabo Hu,Quanhui Ma,Sanqiang Hu,Yanyan Wang,Xiangmei Wen,Yongbin Ma,Hong Xu,Hui Qian,Wenrong Xu
An immortalized mouse fetal liver stromal cell line, named KM3, has demonstrated the potential to support the growth and maintenance of human embryonic stem cells (hESCs). In this study, the characteristics of KM3 cells were examined following cryopreservation at -70˚C and in liquid nitrogen for 15, 30 and 60 days following treatment with 10 µg/ml mitomycin C. In addition, whether the KM3 cells were suitable for use as feeder cells to support the growth of hESCs was evaluated. The inhibition of mitosis without cell death was observed when the KM3 cells were treated with 10 µg/ml mitomycin C for 2 h. The morphology of the KM3 cells cryopreserved in liquid nitrogen for 60 days was not markedly changed, and the cell survival rate was 84.60±1.14%. By contrast, the survival rate of the KM3 cells was 66.40±2.88% following cryopreservation at -70˚C for 60 days; the cells readily detached, were maintained for a shorter time, and had a reduced expression level of basic fibroblast growth factor. hESCs cultured on KM3 cells cryopreserved in liquid nitrogen for 60 days showed the typical bird's nest structure, with clear boundaries and a differentiation rate of 16.33±2.08%. The differentiation rate of hESCs cultured on KM3 cells cryopreserved at -70˚C for 60 days was 37.67±3.51%. These results indicate that the cryopreserved KM3 cells treated with mitomycin C may be directly used in the subculture of hESCs, and the effect is relatively good with -70˚C short-term or liquid nitrogen cryopreservation.
研究领域:
干细胞生物学
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细胞培养
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低温生物学
✓ 已验证
DOI
PubMed
高影响因子
SKIN RESEARCH AND TECHNOLOGY
·
2023
·Vol. 29
·期 2
·e13290
·
IF 2.2
Zhang Mingqi,Wang Le,Zheng Yuqiang,Li Na,He Wei,Wang Zhuoshi
Background and objectives Human epidermal cell sheet (human-ECS) is a feasible treatment option for wound injury. Traditionally, researchers often use murine 3T3 fibroblast cells as feeder layer to support human epidermal cell sheet grafts, thus increase risk to deliver animal-borne infection. To overcome the potential risks involved with xenotransplantation, we develop human foreskin fibroblast cell as feeder layer culture system and investigate the effects of human-ECS on second-degree burn wound healing in mini-pig in order to develop more effective and safer therapies to enhance wound healing in human. Materials and methods Human epidermal keratinocytes and fibroblasts were isolated from foreskin tissue and were co-cultured to manufacture human-ECS. The cell morphology was monitored with phase-contrast microscopy, the stem cell markers were assessed by flow cytometry, and by colony-forming efficiency (CFE) assay. The structure of human-ECS was observed by hematoxylin and eosin staining. Expression of cytokines in human-ECS was confirmed by enzyme-linked immunosorbent assay. Second-degree burn wounds were created on the dorsal of miniature pig to evaluate the effect of oil gauze, oil gauze combined with commercial epidermal growth factor (EGF) cream, and oil gauze combined with human-ECS. Wound healing rate, histological examination, and Masson staining were measured to observe the wound repair efficacy. Real-time PCR and Western blot were utilized to detect the expression level of EGF and interleukin 6 (IL-6). Results Stratified human-ECS with 6-7 layers of epidermal cells was successfully cultivated with human-derived feeder cells, in which epidermal cell highly expressed CD49f and CFE was 3% ± 0.45%. Application of human-ECS induced a higher wound healing rate than commerical EGF cream and oil gauze control. The expression of EGF in human-ECS group was higher than those in the other groups; however, the expression of IL-6 was significantly decreased at day 14 by human-ECS treatment group. Conclusions Human-derived feeder cells are suitable for cultivation of human-ECS, avoiding pathogen transmission. Human-ECS could enhance second-degree burn wound healing, and its promoting effect involved secreting a variety of cytokines to regulate tissue reparative process.
研究领域:
再生医学
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组织工程
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烧伤治疗
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细胞培养
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Nanoscale
·
2014
·Vol. 6
·期 12
·6959-6969
·
IF 6.739
Zhonglin Lyu,Hongwei Wang,Yanyun Wang,Kaiguo Ding,Huan Liu,Lin Yuan,Xiujuan Shi,Mengmeng Wang,Yanwei Wang,Hong Chen
Efficient control of the self-renewal and pluripotency maintenance of embryonic stem cell (ESC) is a prerequisite for translating stem cell technologies to clinical applications. Surface topography is one of the most important factors that regulates cell behaviors. In the present study, micro/nano topographical structures composed of a gold nanoparticle layer (GNPL) with nano-, sub-micro-, and microscale surface roughnesses were used to study the roles of these structures in regulating the behaviors of mouse ESCs (mESCs) under feeder-free conditions. The distinctive results from Oct-4 immunofluorescence staining and quantitative real-time polymerase chain reaction (qPCR) demonstrate that nanoscale and low sub-microscale surface roughnesses (Rq less than 392 nm) are conducive to the long-term maintenance of mESC pluripotency, while high sub-microscale and microscale surface roughnesses (Rq greater than 573 nm) result in a significant loss of mESC pluripotency and a faster undirectional differentiation, particularly in long-term culture. Moreover, the likely signalling cascades engaged in the topological sensing of mESCs were investigated and their role in affecting the maintenance of the long-term cell pluripotency was discussed by analyzing the expression of proteins related to E-cadherin mediated cell–cell adhesions and integrin-mediated focal adhesions (FAs). Additionally, the conclusions from MTT, cell morphology staining and alkaline phosphatase (ALP) activity assays show that the surface roughness can provide a potent regulatory signal for various mESC behaviors, including cell attachment, proliferation and osteoinduction.
研究领域:
干细胞生物学
·
纳米技术
·
细胞工程
·
生物材料科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）