The main factors which affected the isolation, purification and cultivation of Pinellia cordata protoplasts from leaves were studied. The results indicated that the optimum enzyme solution for P. cordata leaves was 13% CPW + 1.0% Cellulose +0.1% Pectolase, at pH 6.0, temperature (25-28 degrees C ) for 4 h. The sucrose density gradient centrifugation was adopted to purificate the protoplasts collected, when 25% sucrose was used as mediator, centrifugating at 500 rpm for 10 min. When the protoplasts were shallow liquid and liquid-solid double layer cultured on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA + 13% mannitol at the density of 2.5 x 104 protoplasts/mL, or fed and nursed cultured at the density of 100-500 protoplasts/mL, cell division could be observed for 3 days; granular calli appeared for 30 days. Calli was proliferated on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA solidified by 0.55% agar, and differentiated and regenerated after 5-6 months. Plant generation of P. cordata is successfully established.
Cell Separation ; methods ; Culture Media ; Pinellia ; physiology ; Protoplasts ; physiology ; Regeneration

Cell Separation ; methods ; Cells, Cultured ; Hepatic Stellate Cells ; cytology ; Humans

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.

Cell Culture Techniques ; methods ; Cell Line, Tumor ; Cell Separation ; methods ; Humans ; Neoplastic Stem Cells ; cytology

OBJECTIVETo separate, cultivate, purify and identify oral carcinoma-associated fibroblasts (CAFs) preliminarily.

METHODSThe primary oral CAFs and normal fibrolasts (NFs) of oral mucosa were obtained by tissue culture. Then cells were dissociated by 0.25% trypsin and purified by curettage method and trypsinization. Morphological characteristics were observed under light microscope and electron microscope. The certain proteins were examined by immunohistochemistry (SP method).

RESULTSThe third passage purified oral CAFs were maintained. The characteristics of shape and growth of the oral CAFs changed significantly comparing to the NFs. Myofilament and electron dense patch were showed in the oral CAFs by electron microscope. The oral CAFs showed negative staining for cytokeratin, and positive staining for vimentin, alpha-smooth muscle actin and matrix metalloproteinases-2.

CONCLUSIONSThere are obvious differences of the morphological characteristics and expression of certain proteins between the CAFs and NFs. The microecology of the oral tumor-host interface might be one of the most important factors affecting the CAFs.

Cell Culture Techniques ; methods ; Cell Separation ; methods ; Fibroblasts ; cytology ; Humans ; Mouth Neoplasms ; pathology

The aim of this study is to establish a method to culture and purify cerebral astrocyte of tree shrew (Tupaia belangeri), a kind of new laboratorial animal which is a relative of primates. Newborn tree shrews were used in this experiment. The cortex of cerebrum was isolated and placed in 4°C for 20 min to injure neurons. The cortical tissue was disaggregated by trypsin digestion. Differential attachment method was used to remove fibroblasts. The mixed culture was rinsed by trypsin (0.005%) solution to remove neurons. Upon reaching 70% confluence, the culture was subjected to static trypsin digestion until a white slice film exfoliated from the bottom of culture bottle. This film, i.e. astrocyte layer, was taken out and cultured, and the third passage was identified by immunocytochemical staining and immunofluorescence with anti-glial fibrillary acidic protein (GFAP) antibody. The result showed the purity of tree shrew astrocytes was more than 98%. Thus the method to culture highly purified astrocyte of tree shrew was successfully established, which would contribute to further study in central nervous system physiology and diseases in this new laboratorial animal.
Animals ; Astrocytes ; cytology ; Brain ; cytology ; Cell Separation ; methods ; Primary Cell Culture ; methods ; Tupaiidae

OBJECTIVETo compare the effect of continuous and discontinuous density gradient centrifugation for purification of human pancreatic islets with COBE 2991 cell processor.

METHODSHuman pancreases were obtained from brain-dead donors and stored in cold UW solution. The connective tissues were removed from the pancreases, and the pancreatic ducts were perfused with a cold enzyme (Liberase). The islets were then separated by gentle mechanical dissociation and purified with discontinuous (10 pancreases) or continuous (8 pancreases) gradients of HCA-Ficoll in COBE 2991 cell processor. Samples were collected in duplicate for determination of the quantity of islets, islet equivalents (IEQ), and the purity.

RESULTSThe weights of the pancreases before and after connective tissue removal and pancreas duct perfusion, and the quantity of islets obtained (including islets quantity of different diameters and total IEQ) after dissociation were not significantly different. Continuous gradient of HCA-Ficoll, compared with discontinuous gradient, resulted in significantly greater final islet quantity (55,000 IEQ vs 206,000 IEQ, P=0.000) and islet purity (58.0%-/+8.0% vs 33.5%-/+10.3%, P=0.000) and also greater number of islets with a diameter lager than 200 microm (P<0.01).

CONCLUSIONContinuous density gradient centrifugation can be more effective than discontinuous gradient in islet purification.

Cell Count ; Cell Separation ; methods ; Centrifugation, Density Gradient ; methods ; Humans ; Islets of Langerhans ; cytology ; Organ Size

OBJECTIVETo explore a new method of isolation and culture of eccrine sweat gland ductal cells from human split-thickness skin graft in vitro.

METHODSHuman split-thickness skin graft which was presented by volunteer (n = 10) was digested with type II collagenase, and then sweat gland duct were isolated from the split-thickness skin graft, primary cultures were incubated at 37 degrees C in humidified atmosphere of 5% CO2, 95% O2. The cultured eccrine sweat gland ductal cells were identified by analysis CEA, CK8, CK18, CK19 antigens expression with flow cytometry, RT-PCR and Western Blot, and by detecting the electrophysiology with whole cell patch clamp technology.

RESULTSThe isolated eccrine sweat gland ductal cells could grow by adhering to the wall, proliferate in vitro after 48 h of adhering to the wall, and confluens after 2 - 4 weeks of adhering to the wall. The FACs analysis showed the expression of CEA was (90.26 +/- 1.12)%, (89.70 +/- 1.43)%, and CK8 was (94.41 +/- 1.84)%, (93.65 +/- 1.63)% in primary cultured sweat gland ductal cells and primary cultured eccrine sweat gland cells, respectively, and there is no significant difference between the two groups (P > 0.05). Immunocytochemistry staining showed CEA, CK8, CK18, CK19 was positive in sweat gland duct cells, RT-PCR revealed that CEA, CK8, CK18 and CK19 gene expression in sweat gland ductal cells, and Western Blot analysis showed the expression of CEA brand, CK8 brand, CK18 brand, and CK19 brand in sweat gland ductal cells, patch clamp indicated that this cells has distinct amiloride sensitive Na(+) channels.

CONCLUSIONSThe cultured human eccrine sweat gland duct cells in vitro display the markers and biological characteristics of sweat gland epithelial lineage, and this method of digest the split-thickness skin graft to get the sweat gland duct cells is better than classical dissect sweat gland under dissect microscope.

Cell Culture Techniques ; methods ; Cell Separation ; methods ; Cells, Cultured ; Humans ; Sweat Glands ; cytology

AIMTo 1) compare post-wash and post-thaw parameters of sperm processed with PureSperm density gradient technique and swim-up method; and 2) test the efficacy of two commonly available density gradient media PureSperm and ISolate.

METHODSThis prospective study used semen specimens from 22 patients. Specimens from nine patients were processed by both PureSperm density gradient and swim-up method. These specimens were then cryopreserved. Thirteen specimens were processed by both PureSperm (40 % and 80 %) and Isolate (50 % and 90 %) double density gradient techniques. The two fractions processed by both PureSperm and swim-up were analyzed for post-wash sperm characteristics. Post-thaw analysis was done after 24 hours. Sperm fractions obtained after processing with PureSperm and ISolate were compared for post-wash sperm characteristics and ROS levels.

RESULTSSpecimens prepared with PureSperm had significantly higher median total motile sperm counts (TMSC) (32.2 x 10(6) vs. 17.6 x 10(6)), recovery rates (69.2 % vs. 50.0 %), and longevity at 4 hours (83.0 % vs. 55.0 %) compared to specimen prepared by swim-up. Post-thaw specimens also had a higher recovery and longevity at 4 hours with PureSperm as compared to the swim-up. Semen specimens processed by PureSperm had significantly higher total sperm count, TMSC, and percentage recovery rates (30.0 % vs. 19.7 %) than ISolate.

CONCLUSIONSemen quality is better preserved in fresh and cryopreserved semen prepared with PureSperm density gradient compared to swim-up. A significant enrichment of sperm is observed with PureSperm compared to ISolate. Higher recovery rates of mature motile sperm obtained after PureSperm sperm preparation may be beneficial for successful ART.

Cell Separation ; methods ; Centrifugation, Density Gradient ; methods ; Cryopreservation ; methods ; Humans ; Male ; Prospective Studies ; Sperm Motility ; Spermatozoa

OBJECTIVETo isolate stem cells from human exfoliated deciduous teeth (SHEDs) and identify their phenotypes and multi-lineage differentiation potential.

METHODSHuman pulp tissue from exfoliated deciduous teeth were dissected and digested to obtain the single cell suspension. The SHEDs selected by magnetic activated cell sorting system (MACS) were identified by examination of the cell morphology and growth in vitro and detection of the expressions of the cell markers. Osteogenic and adipogenic induction was performed to test the multi-lineage differentiation potential of the cells.

RESULTSSHEDs were successfully isolated from human exfoliated deciduous teeth. SHEDs showed a lower growth rate than dental pulp cells and displayed high expressions of CD29 and CD105 but low expressions of CD34 and CD45 as shown by flow cytometry. Experiments of in vitro induction demonstrated a strong potential of the STRO-1+ SHEDs for osteogenic and adipogenic differentiation.

CONCLUSIONImmunomagnetic bead selection can be used to isolate and purify SHEDs, and the STRO-1+ SHEDs show the characteristics of stem cells with multipotent differentiation potentials.

Cell Separation ; Cells, Cultured ; Dental Pulp ; cytology ; Humans ; Immunomagnetic Separation ; methods ; Stem Cells ; cytology ; Tooth, Deciduous ; cytology

A protocol for enrichment and adsorption of karyocyte from whole blood by using magnetic nanometer beads as solid-phase absorbents was presented. The PCR amplification could be accomplished by using the nanobeads with karyocyte as template directly and the PCR products were applied on an oligonucleotide array to do gene typing. The HLA-A PCR amplification system and a small HLA-A oligonucleotide microarray were applied as the platform and an experiment protocol of separating karyocyte from whole blood using the magnetic nanometer beads (Fe2O3) were set up. The experimental conditions were also discussed. It showed that pH level of PBS eluent, Taq enzyme quantity and fragment length of products could influent the amplification results, and the magnetic nano-beads could succeed in sample preparation in microarray to provide a promising way in automatic detection and lab-on-a-chip.
Adsorption ; Cell Separation/instrumentation ; Cell Separation/methods ; Genotype ; Leukocytes/*cytology ; *Magnetics ; Microchemistry/instrumentation ; Microchemistry/*methods ; Nanotechnology ; Oligonucleotide Array Sequence Analysis/*methods ; Polymerase Chain Reaction ; Templates, Genetic