To evaluate the maturation and differentiation state of cultured keratinocytes, the author investigated expression of differentiation markers in cultured keratinocytes. The specimens were divided into three experimental groups, 3rd passage keratinocytes cultured in serum free media (3rd SFM group), 6th passage keratinocytes cultured in serum free media (6th SFM group) and 3rd passage keratinocytes cultured in DMEM (DMEM group). CK14, marker of basal layer, expressed in all groups. The expression was localized and condensed in the SFM groups but spreade in the DMEM group. Most of the cells in both SFM groups were positive but a few cells in DMEM group were also positive. CK10, marker of initiation of differentiation, expressed weakly in DMEM group but there was no expression in both SFM groups. Involucrin, marker of terminal differentiation, expressed weakly in DMEM group but there was no expression in both SFM groups. CK16 and 17, markers of fast turnover of keratinocytes, were not expressed in SFM groups. Weak positive reactions were observed in DMEM group. With these results the authors concluded that the keratinocytes from 3rd passage to 6th passage, cultured in serum free media with calcium less than 0.1 mM, had highly homogeneous basal cell characteristics.
Antigens, Differentiation ; Calcium ; Culture Media, Serum-Free ; Humans* ; Keratinocytes* ; Keratins*

Insect cell-baculovirus system is a useful tool for both insecticidal virus production and the expression of medically useful foreign genes. Serum-free culture or low serum culture for insect cells is essential and significant. Media for the growth of insect cells HzAm1 from three kinds of commercial media TC-100,GRACE and IPL-41 with 10% serum were investigated and screened. The result shows that medium TC-100 is the most suitable medium for the growth of cells HzAm1. Adaptation of insect cells HzAm1 to cultures in medium TC-100 with serum reduction from 10% to 1% was carried out as cultures were supplemented with lactalbumin hydrolysate and yeastolate etc. Growth of insect cells HzAml in medium TC-100 with serum 1%, lactalbumin hydrolysate and yeastolate was well.
Adaptation, Physiological ; Animals ; Cell Proliferation ; Culture Media ; Culture Media, Serum-Free ; Lepidoptera ; cytology

culture processes for viral vaccine production are mainly based on adherent cell culture systems using serum, which are associated with expensive and labor-intensive processes to produce large amounts of viral vaccine strains. In this study, we investigated whether Vero cells could be grown in serum-free and shaking suspension conditions. Furthermore, we assessed the ability of the Vero cell suspension culture system to produce adenovirus type 5 (Ad5), compared to that of the adhesive Vero cell culture system.MATERIALS AND METHODS: We tested the feasibility of commercial serum-free media for Vero cell culture. For the adaptation of Vero cells in suspension culture, adhesive Vero cells were added in the early phase of shaking suspension culture, and 50 days after shaking suspension culture, suspension-adapted Vero cells were subcultured continuously. To assess the virus production ability of Vero cells in suspension, the cells were infected with Ad5-green fluorescent protein and evaluated based on their fluorescence intensity.RESULTS: The Vero cells grown in OptiPRO serum-free medium showed no changes in morphology and growth rate, but MRC-5 and FRhk-4 cells showed morphological changes and decreased growth rate, respectively. The Vero cells were well adapted to the suspension culture system. The Vero cells in suspension showed a better Ad5 production ability than the adherent Vero cells.CONCLUSION: Vero cells can be grown in OptiPRO serum-free medium. Further, our suspension culture-adapted Vero cells may be suitable to produce viral vaccine strains due to their high ability to produce viruses such as Ad5.]]>
Adenoviridae ; Adhesives ; Cell Culture Techniques ; Culture Media, Serum-Free ; Fluorescence ; Vero Cells

BACKGROUND: Research on RBC production from hematopoietic stem cells has been conducted competitively in many countries. However those were in vitro successes and many hurdles still remain for large scale transfusable RBC production from stem cells. A need for large volume of culture media is a crucial factor for culture condition which researchers must overcome. In this study, we evaluated the efficiency of two commercial serum-free media, StemPro(R)-34 SFM and Stemline II hematopoietic stem cell expansion medium, in RBC differentiation from cord derived stem cells. METHODS: We cultured cord derived CD34+ cells in vitro and evaluated over the periods of 7 days, 14 days, 17 days and 21 days in culture for expanded cell count, cell morphology and differential count using the Wright Giemsa stain. RESULTS: Cell expansion and RBC differentiation developed rapidly in Stemline media compared to StemPro media. Enucleated RBCs were observed at 10~14 culture days and orthochromatic erythroblasts were shown up to 50% among culture cells at 17 days in Stemline media. The enucleated RBCs were observed at 17 days in StemPro Media. Although the erythroblasts in StemPro media are slow at differentiation, they maintain continuous expansion up to 21 days. CONCLUSION: In Stemline media, the expansion and differentiation to mature RBCs are processed much faster, but the cell condition slows down after 17 days. In the RBC production aspects, Stemline media is better than StemPro media as a rapid differentiation because it reduces the cost due to in vitro short culture duration.
Azure Stains ; Cell Count ; Culture Media ; Culture Media, Serum-Free* ; Erythroblasts ; Hematopoietic Stem Cells* ; Humans ; Stem Cells

PURPOSE: To evaluate the effect of nitric oxide (NO) on the survival of R28 cells. METHODS: After immunostaining for GFAP, vimentin, S-100, and neurofilament, R28 cells were exposed to S-nitroso-N-acetyl-D, L-penicillamine (SNAP) at various concentrations, with and without the NO inhibitor, Nomega-Nitro-L-arginine methyl ester (L-NAME) for 1 and 3 days. Cellular survival of R28 cells and the production of NO were quantified by rapid colorimetric assays using the MTT and Griess assay, respectively. To evaluate the effect of serum, 10% serum or serum-free media were used separately. RESULTS: R28 cells showed strong immunoreactivity to GFAP and vimentin compared to S-100 or neurofilament. SNAP inhibited the survival of R28 cells in a dose-dependent manner, and this effect of NO on the cellular survival was abolished by L-NAME. These results were similar after exposure for 1 and 3 days, regardless of the presence of serum in the media. CONCLUSIONS: The current results suggest that NO decreased the survival of R28 cells. Further studies are necessary to evaluate the mechanism of cytotoxicity of the R28 cells.
Culture Media, Serum-Free ; NG-Nitroarginine Methyl Ester ; Nitric Oxide ; Vimentin

PURPOSE: To evaluate the protective effect of creatine on the survival of retinal ganglion cells after serum deprivation. METHODS: RGC-5 cells were exposed to 5 mM creatine with serum-free media for 4 days. Cellular survival and mitochondrial respiratory activity were measured with MTT assay and resazurin assay, respectively. Degree of apoptosis was evaluated with vital staining using acridine orange/Hoechest 33342 and flow cytometric analysis using annexin/PI, respectively. RESULTS: Creatine increased cellular survival of RGC-5 cells significantly after serum deprivation. Additionally, creatine increased mitochondrial respiratory activity and inhibited apoptosis of RGC-5 cells. CONCLUSIONS: The energy precursor creatine increased survival of retinal ganglion cells after serum deprivation. Creatine could be relevant for the cytoprotection of retinal ganglion cells.
Apoptosis ; Creatine ; Culture Media, Serum-Free ; Cytoprotection ; Oxazines ; Retinal Ganglion Cells ; Xanthenes

The importance of recombinant adenoviral vectors for the development of gene therapy and prophylactic and therapeutic vaccines has led to efforts for process development of large scale production of clinically safe adenoviral vectors. First of all, cell lines producing replication incompetent adenoviral vectors required for clinical application have been developed and the concept of banking and characterization of cell lines and adenoviral vectors has been established. In order to meet the need of amount of adenoviral vectors for clinical trials, various large scale suspension culture methods using serum-free media have been developed along with development of large scale purification methods using chromatography instead of cesium chloride method. In addition, methods for the quality control of adenoviral vectors have been established and applied for the clinical lots.
Cell Line ; Cesium ; Chlorides ; Chromatography ; Culture Media, Serum-Free ; Genetic Therapy ; Quality Control ; Vaccines

It is the aim of this study to investigate the effects of sodium fluoride and sodium orthovanadate upon the proliferation and activity of the osteoblast (MC3T3-E1 cells). MC3T3-E1 cells were cultured in alpha-MEM containing 10% FBS and various concentration of sodium fluoride and sodium orthovanadate was appended to serum free media. DNA synthesis was examined through the [3H] thymidine incorporation into DNA. Collagen synthesis was examined through the [3H] proline incorporation into collagenase digestible protein and noncollagen protein. The following results were drawn; 1. Sodium fluoride stimulated the DNA synthesis of osteoblast significantly in dose-dependent manner within the concentration from 2micrometer to 10micrometer (P<0.005). 2. Sodium orthovanadate stimulated the DNA synthesis of osteoblast significantly in dosedependent manner within the concentration from 2micrometer to 8micrometer, however showed diminution at 10micrometer (P<0.001). 3. Sodium fluoride and sodium orthovanadate stimulated the percent collagen synthesis of osteoblast significantly in dose-dependent manner within the concentration from 5micrometer to 10micrometer (P<0.001). 4. Sodium fluoride and sodium orthovanadate stimulated the non collagen synthesis of osteoblast significantly in dose-dependent manner within the concentration from 5micrometer to 10micrometer (P<0.001). In conclusion, sodium fluoride and sodium orthovanadate stimulate the proliferation and activity of osteoblast by stimulation of DNA synthesis and collagen and noncollagen synthesis in osteoblast.
Cell Line* ; Collagen ; Collagenases ; Culture Media, Serum-Free ; DNA ; Osteoblasts* ; Proline ; Sodium Fluoride* ; Sodium* ; Thymidine ; Vanadates*

OBJECTIVE: To compare the efficacy of directional erythroid differentiation in different serum free culture systems and to screen the optimal culture systems for inducing the differentiation of umbilical cord blood hematopoietic stem and progenior cells (HSPC) to erythroid cells. METHODS: The CD34 cells from umbilical blood munonuclear cells were sorted by using the magnetic beads, and were inoculated into 3 different of culture systems (system 1, 2 and 3 respectively), to induce erythrold differentiation by 3 stage culture. The living cells were counted in different differentiation stages and were observed by Wright-Giemsa staining; the expression of CD71 and CD235a on cell surface was detected by flow cytometry, the erythroid differentiation pteency was detected via colony-forming test. RESULTS: The ability of system 2 to promote the HSPC proliferation was the strongest, the efficacy of system 3 to promote the erythroid differentiation of HSPC was the most optimal; the proliferation ability of cells cultured in system 2 for 2-15 days all was higher than that of cells cutured in system 1 and 3 (P＜0.05). The flow cytometry detection showed that the expression of CD71 and CD235a on surface of cells cultured in system 3 was the highest, the CD235a percentage on day 15 of differentiation in system 3 was (92.33±3.89)%, that in system 2 was (84.67±3.12)%, while that in system 1 was (72.17±6.83)% (P＜0.05). Cell morplologic detection showed that throid differentiation was accelerated on day 12, the percentage of orthochromatic erythrocytes in system 3 was (67.67±2.08)% which was 10.69 and 25.34 times higher than that in system 2 and 1 respectively (P＜0.05). The colony-forming test showed the ratio of BFU-E in system 3 increased gradually on day 3-9 (r=0.99, P＜0.05), which was significanlly higher than that in system 2 and 1 on day 9 (90.35±5.52% vs 77.06±2.26% and 74.50±3.95%). CONCLUSION Culture system 3 is the most effective serum-free erythroid differentiation system, and the culture system 2 is the most powerful HSPC proliferation system. This study results provide a technical basis for further efficiently increasing and inducing the erythroid proliferation and differentiation of HSPC, and also provide culture system in vitro for the clinical application and basic research.
Antigens, CD34 ; Cell Differentiation ; Cells, Cultured ; Culture Media, Serum-Free ; Erythroid Precursor Cells ; Fetal Blood ; Humans

OBJECTIVETo establish an optimal method for serum-free and feeder layer-free culture of human keratinocytes and to investigate their biological characteristics.

METHODSThe keratinocytes were harvested from human foreskin of 5 children (aged 5-10 yr) and 5 adults (aged 20-30 yr). The samples were isolated by two-step digestion and the quantities of primary harvested HKCs were determined. The HKCs were then cultured in KCS serum-free culture medium. The morphology of HKCs were observed under light microscope. The HKCs and their growing speed were observed and identified under fluorescent microscope. The growth curve of HKCs was detected with MTT method, and the cell cycle was determined with flow cytometry.

RESULTSThe number of harvested HKCs from children [(1.780 +/- 0.010) x 10(6)/cm(2)] was obviously higher than that from adults [(1.490 +/- 0.120) x 10(6)/cm(2)], (P < 0.01). Freshly isolated primary HKCs were round and transparent, and 94% of them were trypan blue resistant. The adherent speed and rate and lucent degree of multiply passaged HKCs increased followed by each passage. Under the fluorescent microscope, the cells exhibited strong Kelly fluorescence in the cytoplasm and with no staining in the nucleolus, thus the cells were identified as HKCs. The HKCs from children for skin could be passaged for more times [(11.0 +/- 1.2) times] than that from adults [(9.2 +/- 0.8) times], (P < 0.05). There was no clear sign of incubation period in the growth curve of HKCs, and both cellular proliferating speed and rate of proliferation were high. The percentage of cells in G1, G2 and S phase and the proliferation index was 36.15%, 25.17%, 38.68% and 63.85%, respectively.

CONCLUSIONSerum-free and feeder layer-free culture seems to be an ideal method for the cultivation of HKCs.

Adult ; Cell Culture Techniques ; methods ; Cells, Cultured ; Child ; Child, Preschool ; Culture Media, Serum-Free ; Humans ; Keratinocytes ; cytology ; Male ; Young Adult