OBJECTIVETo acquire lots of cell to culture during the primary cell culture.

METHODWe take the split-thickness skin slide technique to acquire the dissociated fibroblast cell in two big-ear rats.

RESULTSThe cell number is above 10(6) from 1 cm x 2 cm split-thickness skin slide and the technique is simple, economic, effectve.

CONCLUSIONWe think this way is better than other methods, and should be adopted in the primary cell culture, especially in fibroblast transplantation by injection.

OBJECTIVE: This study optimizes three-dimensional (3D) culture conditions of HepG2 using response surface methodology (RSM) based on the VitroGel system to facilitate the cell model in vitro for liver tissues. METHOD: HepG2 cell was 3D cultured on the VitroGel system. Cell viability was detected using Cell Counting Kit-8 (CCK-8) assay of HepG2 lived cell numbers. The proliferation of HepG2 cell and clustering performance was measured via fluorescence staining test. Albumin concentration in the culture medium supernatant as an index of HepG2 cell biological function was measured with ELISA kit. Independent factor tests were conducted with three key factors: inoculated cell concentration, cultured time, and dilution degree of the hydrogel. The preliminary results of independent factor tests were used to determine the levels of factors for RSM. RESULT: The selected optimal culture conditions are as follows: concentration of inoculated cells was 4.44 × 10 ⁵/mL, culture time was 4.86 days, and hydrogel dilution degree was 1:2.23. The result shows that under optimal conditions, the predicted optical density (OD) value of cell viability was 3.10 and measured 2.978 with a relative error of 3.94%. CONCLUSION This study serves as a reference for the 3D HepG2 culture and constructs liver tissues in vitro. Additionally, it provides the foundation for repeated dose high-throughput toxicity studies and other scientific research work.
Albumins ; Cell Culture Techniques/methods* ; Hep G2 Cells ; Humans ; Hydrogels

As the cell co-culture techniques can better imitate an in vivo environment, it is helpful in observing the interactions among cells and between cells and the culture environment, exploring the effect mechanisms of drugs and their possible targets and filling the gaps between the mono-layer cell culture and the whole animal experiments. In recently years, they has attracted much more attention from the medical sector, and thus becoming one of research hotspots in drug research and development and bio-pharmaceutical fields. The cell co-culture techniques, including direct and indirect methods, are mainly used for studying pathological basis, new-type treatment methods and drug activity screening. Existing cell co-culture techniques are used for more pharmacological studies on single drug and less studies on interaction of combined drugs, such as collaborative compatibility and attenuation and synergistic effect among traditional Chinese medicines (TCMs). In line with the action characteristics of multi-component and multi-target, the cell co-culture techniques provide certain reference value for future studies on the effect and mechanism of combined TCMs on organisms as well as new methods for studies on TCMs and their compounds. This essay summarizes cell co-culture methods and their application and look into the future of their application in studies on TCMs and compounds.
Animals ; Biomedical Research ; methods ; Cell Culture Techniques ; methods ; Coculture Techniques ; methods ; Humans

Microfluidic chip is a novel technology platform, in which microchannels are fabricated in different materials. The ability to precisely control the microflows makes it possible to mimic the microenvironment of cells in physiological or pathological states, which provides many distinct advantages for cell research. In this paper are reviewed the design and fabrication of microfluidic chip, the application of microfluidic chip in cell culture and cell researches; the enormous advantages of microfluidic chips in precise experimental control of the cellular microenvironment are introduced.
Cell Adhesion ; Cell Culture Techniques ; Cell Movement ; Cells, Cultured ; Cellular Microenvironment ; Humans ; Microfluidic Analytical Techniques ; methods

In this article, a cell culture microchip was fabricated on the SU-8 mold based on polymer-MEMS process. In the microchip, the cell culture area was separated with microchannel by a microgap, which kept the cell culture area independent, but also regulated the micro-environment of extracellular matrix by the microfluidic flow. The cell culture microchip provided a new platform for cell research.
3T3 Cells ; Animals ; Cell Culture Techniques ; methods ; Mice ; Microfluidic Analytical Techniques ; methods

OBJECTIVETo develop an effective method for isolating and culturing single cancer stem cells.

METHODSThe capillary glass tube was stretched on fire and connected to a sterile plastic tube to prepare the single cell separation apparatus. Single SW480 cell clone spheres in serum-free culture were marked with CD133 and CK7, and the single cancer stem cells were separated and cultivated in 96-well plates or microdrop covered by paraffin.

RESULTSSW480 cell clone formation rate was about 1.04%, and the cell clone spheres highly expressed CD133 with low CK7 expression. The isolation of the single cancer stem cells showed a success rate of 98.99% using the separation device. The cell division profile was comparable between the cell cultures in microdrop and 96-well plates in the initial 2 cell divisions (P>0.05), whereas prolonged cell division occurred afterwards in the microdrop culture as compared to 96-well plate culture. The cell population expansion of the single cancer stem cells was similar between microdrop culture (11.5%, 22/192) and 96-well plate culture (9.2%, 17/184) (P>0.05).

CONCLUSIONSSingle SW480 cells can develop into cancer stem cell spheres. Microdrop culture is convenient and stable, and can be the primary choice for single cancer stem cell culture.

In this research, we use mouse embryonic fibroblasts as feeder layers. To eliminate the influence of serum and mouse embryonic stem cells (ESCs) conditioned medium (ESCCM) on self-renewal of sheep embryonic stem-like cells, knockout serum replacement (KSR) was used to replace serum, then supplanted with ESCCM for the isolation and cloning of sheep embryonic stem-like cells. We found when inner cell masses (ICMs) cultured in the control group with medium supplanted with fetal bovine serum (FBS), sheep ES-like cells could not survive for more than 3 passages. However, sheep embryonic stem-like cells could remain undifferentiated for 5 passages when cultured in the medium that FBS was substituted by KSR. The result indicates that KSR culture system was more suitable for the isolation and cloning of sheep embryonic stem-like cells compared to FBS culture system. Finally we applied medium with 15% KSR as basic medium supplanted with 40% ESCCM as a new culture system to isolate sheep embryonic stem-like cells, we found one embryonic stem-like cell line still maintained undifferentiating for 8 passages, which characterized with a normal and stable karyotype and high expression of alkaline phosphatase. These results suggest that it is suitable to culture sheep ICM in the new culture system with 15% KSR as basic medium and supplanted with 40% ESCCM, which indicated that mouse ES cells might secrete factors playing important roles in promoting sheep ES-like cells' self-renewal.
Animals ; Cell Culture Techniques ; methods ; Cell Proliferation ; Cell Separation ; methods ; Clone Cells ; Culture Media ; Embryonic Stem Cells ; cytology ; Mice ; Sheep

The extreme microorganisms Ferroplasma spp., play an important role in bioleaching of sulphide ores at low pH value and temperatures around 50 degrees C. Without cell wall, Ferroplasma spp. is sensitive to pulp density, shearing force and heavy metal ions. Thus it is difficult to obtain their high cell density cultures, which limits the large-scale industrial application. In this paper, the optimum culture conditions of Ferroplasma thermophilum were studied by shaking culture. The results showed that the optimum culture conditions are as follows: 50 degrees C, initial pH 0.5, 50 mL working volume in 250 mL shaking-flask, inorganic nitrogen source (NH4)2SO4. The optimum combination of FeSO4.7H2O, yeast extract and peptone was determined by orthogonal experiments, including FeSO4.7H2O 40 g/L, yeast extract 0.3 g/L, peptone 0.2 g/L. Under the optimum culture conditions, the cell density was up to 6.3 x 10(7) cell/mL, and the oxidation of 40 g/L ferrous sulfate heptahydrate was finished in less than 72 hours. The results might provide information for scale-up of archaeon culture as well as its industrial application.
Archaea ; cytology ; growth & development ; Cell Culture Techniques ; methods ; Culture Media ; Hydrogen-Ion Concentration ; Metallurgy ; Temperature

The effects of the cultivation media, plant growth regulators and inoculum size on the cell growth and 20-hydroxyecdysone production in suspension cultures of Vitex glabrata R. Br. were investigated. The cell growth and 20-hydroxyecdysone formation reach the highest when cells are cultured in the Gamborg's B5 medium supplemented with 2.0 mg/L BAP (6-benzylaminopurine) and 1.0 mg/L 2,4-D. The maximum 20-hydroxyecdysone productivity, of about 1.l mg/L/day, was observed in the culture with 20% PCV (packed cell volume) of inoculum size. These data also show that the increment of the inoculum size to 20% PCV could increase the productivity in 7-folds.
Cell Culture Techniques ; methods ; Culture Media ; Ecdysterone ; biosynthesis ; Vitex ; cytology ; metabolism

Animals ; Cell Culture Techniques ; methods ; Cell Separation ; methods ; Culture Media ; Epithelial Cells ; cytology ; Respiratory System ; cytology ; Swine