BACKGROUND: Psoriasis is a common, scaly erythematous disease of unknown etiology, marked by remissions and exacerbations of unpredictable onset and duration. Among many etiologic factors, psoriatic keratinocyte is found to play the most important role. OBJECTIVE: The purpose of this study is to evaluate the hypothesis that the mechanism(s) responsible for the abnormal proliferation of psoriatic keratinocytes may be located within the cell themselves. METHODS: Human epidermal keratinocytes were isolated from lesion(PL) and from uninvolved skin (PN) with chronic plaque-like psoriasis and from the normal skin(NN). Keratinocytes were passaged onto culture vessels without the feeder layer and maintained with serum free medium. Growth rates were measured in secondary cultures by MTT assay and ultrastructural findings of cell differentiation were evaluated with a transmission electron microscope. Results : 1 Keratinocytes from PL reached 50% confluency in one week compared to two weeks of PN and NN in primary cultures. 2. By the MTT assay, keratinocyte proliferation from PL showed a significantly faster rate than those from PN and NN(p<0.01). But there was no significant difference of keratinocyte proliferation rate between PN and NN(p>0.05). 3. All of the three cell populations(PL, PN, NN) showed variable degrees of cell differentiation during secondary culturing in a serum-free medium. In the PL, however, small, compact basal cells were more prevalent than PN and NN. 4. When keratinocytes underwent differentiation by culturing in DMEM with serum, keratinocytes from PL formed more cell layers with incomplete formation of cornified envelopes suggests the presence of some unknown factors that induce or promote psoriasis. While keratinocytes from PN and NN were characterized by a complete codified layer as in normal skin. Conclusion : These results indicated that the characteristic hyperproliferation and the defective terminal differentiation of keratinocytes of PL were maintained throughout the culture period.
Cell Differentiation ; Feeder Cells ; Humans ; Keratinocytes* ; Psoriasis ; Skin

This study was performed to observe the influence of the feeder layer on the culture of the human uveal melanoma cell. Two types of melanoma cells which were obtained from the patients with choroidal melanoma were implanted into the culture flask. The melanoma cell was seeded onto the culture dish covered with conjunctival fibroblast, Vero cell, and culture dish which did not contain the feeder layer respectively. The growth pattern of the melanoma cell was observed with phase contrast microscopy upto 30 days after seeding. There was no definite difference in growth pattern between each group. These results may indicate that the feeder layer is not an essential factor in the culture of uveal melanoma cell.
Choroid ; Feeder Cells ; Fibroblasts ; Humans ; Melanoma* ; Microscopy, Phase-Contrast ; Vero Cells

OBJECTIVE: This study was carried out to evaluate the effect of the isolation methods of inner cell mass from mouse blastocyst, types of feeder cells and treatment time of mitomycin C on the formation rate of ICM colony. METHODS: The inner cells were isolated by conventional immunosurgery, partial trophoblast dissection with syringe needles and whole blastocyst co-culture method. Commercially available STO and primary cultured mouse embryonic fibroblast (pMEF) feeder cells were used, and mitomycin C was treated for 1, 2 or 3 hours, respectively. The formation rate of ICM colony was observed after isolation of ICM and culture of ICM on the feeder cells for 7 days. RESULT: The ICM colony formation rate on STO were significantly higher in partial trophoblast dissection group (58%) than that in immunosurgery (12%) or whole blastocyst culture (16%) group (p<0.05). The formation rate on pMEF feeder layer was higher in partial trophoblast dissection (88%) and whole blastocyst culture (82%) group than that in immunosurgery (16%) group (p<0.05). When mitomycin C treated to pMEF for 2 hours, the formation rate of 88% was significantly higher than those of other conditions. CONCLUSIONS: Above results showed that the efficient isolation method of ICM from blastocyst was the partial trophoblast dissection and the appropriate treatment time of mitomycin C was 2 hours. However, the subculture of ICM colony and characterization of stem cells should be carried out to confirm the efficacy of the partial trophoblast dissection method.
Animals ; Blastocyst ; Coculture Techniques ; Feeder Cells* ; Fibroblasts ; Mice* ; Mitomycin* ; Needles ; Stem Cells ; Syringes ; Trophoblasts

BACKGROUND: Natural killer (NK) cells constantly survey surrounding tissues and remove newly generated cancer cells, independent of cancer antigen recognition. Although there have been a number of attempts to apply NK cells for cancer therapy, clinical application has been somewhat limited because of the difficulty in preparing a sufficient number of NK cells. Therefore, ex vivo NK cell expansion is one of the important steps for developing NK cell therapeutics. METHODS: CD3+ depleted lymphocytes were cocultured with IL-2 and with feeder cells (peripheral blood mononuclear cells [PBMCs], K562, and Jurkat) for 15 days. Expanded NK cells were tested for cytotoxicity against cancer cell lines. RESULTS: We compared feeder activities of three different cells-PBMC, K562, and Jurkat. K562 expanded NK cells by almost 20 fold and also showed powerful cytotoxic activity against cancer cells. K562-NK cells remarkably expressed the NK cell activation receptors, NKG2D, and DNAM-1. K562-NK cells exhibited more than two-fold production of cytotoxic granules compared with Jurkat-NK cells, producing more perforin and granzyme B than naive NK cells. CONCLUSION: Our findings suggest that K562 are more efficient feeder cells than Jurkat or PBMCs. K562 feeder cells expanded NK cells by almost 20 fold and showed powerful cytotoxic activity against cancer cells. We herein propose an intriguing approach for a design of NK cell expansion.
Cell Line* ; Feeder Cells* ; Granzymes ; Humans ; Interleukin-2 ; Killer Cells, Natural* ; Leukemia, Myeloid* ; Lymphocytes ; Perforin

Recovery from potentially lethal damage (PLDR) after irradiation was studied in plateau-phase culture of Vero cells in vitro. Unfed plateau-phase cells were irradiated with dose of 1 to 9 gy using Cs-137 irradiator. Cells then were incubated again and left in situ for 0, 1, 2, 3, 4, 5, 6 and 24 hours and then were trypsinized, explanted, and subcultured in fresh RPMI-1640 media containing 0.33% agar. Cell survival was measured by colony forming ability. An adequate number of heavily irradiated Vero cells were added as feeder cells to make the total cell number constant in every culture dish. As the postirradiation in situ incubation time increased, surviving fraction increased saturation level at 2 to 4 hours after in situ incubation. As the radiation dose increased, the rate of PLDR also increased. In analysis of cell survival curve fitted to the linear-quadratic model, the linear inactivation coefficient (alpha) decreased largely and reached nearly to zero but the quadratic inactivation coefficient (beta) increased minimally by increment of postirradiation in situ incubation time. So PLDR mainly affected the damage expressed as alpha. In the multitarget model, significant change was not obtained in D0 but in Dq. Therefore, shoulder region in cell survival curve was mainly affected by PLDR and terminal slope was not influenced at all. And dose-modifying factor by PLDR was relatively higher in shoulder region, that is, in low dose area below 3 gy.
Agar ; Cell Count ; Cell Survival ; Feeder Cells ; Shoulder ; Trypsin ; Vero Cells*

OBJECTIVES: Experimental models are of importance to study the pathogenesis of middle ear cholesteatoma, however, they were not established until now. We aimed to develop in vitro model of middle ear cholesteatoma using primary keratinocytes and fibroblasts isolated from cholesteatoma tissue. HaCaT cell line was used as a "skin equivalent" and to compare the grade of homogeneity between cholesteatoma keratinocytes and HaCaT cells. METHODS: Primary keratinocytes were isolated from cholesteatoma tissue, co-cultured with preliminary prepared feeder layer from cholesteatoma fibroblasts and subsequently air-exposed. The protein profile of cholesteatoma keratinocytes and HaCaT cells was evaluated by means of immunoblot using monoclonal antibody against cytokeratin (CK) 13 and 16. Tissue localization of CK 13 and 16 was accomplished with immunohistochemistry. RESULTS: Different protein profile and stronger expression of CK 13 and 16 were demonstrated in cholesteatoma keratinocytes in comparison with HaCaT cells. Bigger stratification was observed in the 3D-in vitro systems when both cholesteatoma keratinocytes and HaCaT cells were respectively co-cultured with fibroblasts in comparison with the corresponding control groups without fibroblasts. CONCLUSION: 3D-model demonstrates the significance of intercellular interaction between components of cholesteatoma tissue.
Cell Line ; Cholesteatoma ; Cholesteatoma, Middle Ear ; Feeder Cells ; Fibroblasts ; Immunohistochemistry ; Keratinocytes ; Keratins ; Models, Theoretical

OBJECTIVE: This study was performed to evaluate the possibility of prolonged culture of human embryonic stem cells (hESC; SNUhES2) on human amniotic fluid cells (hAFC), which had been storaged after karyotyping. METHOD: The hAFC was prepared for feeder layer in the presence of Chang's medium and STO medium (90% DMEM, 10% FBS) at 37degrees C in a 5% CO2 in air atmosphere. Prior to use as a feeder layer, hAFC was mitotically inactivated by mitomycin C. The hESCs on hAFC were passaged mechanically every seven days with ES culture medium (80% DMEM/F12, 20% SR, bFGF). RESULTS: The hAFC feeder layer support the growth of undifferentiated state of SNUhES2 for at least 59 passages thus far. SNUhES2 colonies on hAFC feeder appeared slightly angular and flatter shape as compared with circular and thicker colonies observed with STO feeder layer and showed higher level with complete undifferentiation in seven days. Like hESC cultured on STO feeders, SNUhES2 grown on hAFC expressed normal karyotype, positive for alkaline phosphatase activity, high telomerase activity, Oct-4, SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81 and formed embryoid bodies (EBs). CONCLUSION: The hAFC supports undifferentiated growth of hESC. Therefore, these results may help to provide a clinically practicable method for expansion of hESC for cell therapies.
Alkaline Phosphatase ; Amniotic Fluid* ; Atmosphere ; Embryoid Bodies ; Embryonic Stem Cells* ; Feeder Cells ; Female ; Humans* ; Karyotype ; Karyotyping ; Mitomycin ; Telomerase

OBJECTIVE: To establish the STO cell lines expressing green fluorescent protein (GFP) and mouse leukemia inhibitory factor (LIF) , and try to culture the mouse embryonic stem cells (mESCs) by using the established STO-GFP-mLIF cells as the feeder layer. METHODS: The lentiviral particles containing GFP and mLIF and puromycin-resistance gene were constructed and transduced into STO cell lines. The cell lines stably expressing GFP and mLIF genes were screened out. The expression level of the inserted exogenous LIF gene was tested by Western blot and ELISA. The STO-GFP-mLIF cells were treated with different concentrations of mitomycin C (5, 10, 15, 20 µg/ml) for different time (1.5, 2.5, 3, 3.5 hours) to prepare feeder layers and the cell proliferation level on feeder layer was observed. Mouse embryonic stem cells were cultured on mitomycin C-treated feeder layer and the growth of cell colonies was observed. RESULTS: The expression level of LIF protein in STO-GFP-mLIF cells was up-regulated, as compared with STO cells (P＜0.05). It was confirmed that the optimal concentration and time for inhibiting the proliferetion of STO-GFP-mLIF cells by mitomycin C were 10 µg/ml and 3 hours respectively. The observation also found that the embryonic stem cells could develop into typic "birdnest" shaped stem cell colony on mitomycin C-treated feeder layer. CONCLUSION The stable STO cell lines effectively expressing green fluorescent protein and mouse leukemia inhibitory factor have been established successfully, which can maintain the undifferentiated state of mouse embryonic stem cells.
Animals ; Cell Differentiation ; Cell Line ; Cell Separation ; Embryonic Stem Cells ; Feeder Cells ; Green Fluorescent Proteins ; Leukemia Inhibitory Factor ; Mice

Previously, the majority of human embryonic stem cells and human induced pluripotent stem cells have been derived on feeder layers and chemically undefined medium. Those media components related to feeder cells, or animal products, often greatly affect the consistency of the cell culture. There are clear advantages of a defined, xeno-free, and feeder-free culture system for human pluripotent stem cells (hPSCs) cultures, since consistency in the formulations prevents lot-to-lot variability. Eliminating all non-human components reduces health risks for downstream applications, and those environments reduce potential immunological reactions from stem cells. Therefore, development of feeder-free hPSCs culture systems has been an important focus of hPSCs research. Recently, researchers have established a variety of culture systems in a defined combination, xeno-free matrix and medium that supports the growth and differentiation of hPSCs. Here we described detailed hPSCs culture methods under feeder-free and chemically defined conditions using vitronetin and TeSR-E8 medium including supplement bioactive lysophospholipid for promoting hPSCs proliferation and maintaining stemness.
Animals ; Cell Culture Techniques ; Embryonic Stem Cells ; Extracellular Matrix ; Feeder Cells ; Human Embryonic Stem Cells ; Humans ; Induced Pluripotent Stem Cells ; Pluripotent Stem Cells ; Stem Cells

Gradual attrition of telomere to a critical short length elicits successive cellular response of cellular senescence and crisis. Cancer cells evade this process by maintaining functional telomeres via one of two known mechanisms of telomere maintenance. The first and most frequent mechanism involves reactivation of enzyme activity of telomerase, a ribonucleoprotein complex mainly via transcriptional up-regulation of TERT, a catalytic subunit of telomerase complex. The second mechanism utilizes telomerase-independent way termed ALT (for Alternative Lengthening of Telomere), which possibly involves recombination pathways. Thus master key for cellular immortalization is supposed to possess adequate telomere reserves. Indeed, telomerase can alone induce the immortalization under culture on feeder cell layers without generally known inactivation mechanism of tumor suppressor genes. Including this phenomena, this review will focus on telomerase and telomere-associated proteins, thereby implication of these proteins for cellular immortalization processes.
Carcinogenesis ; Catalytic Domain ; Cell Aging ; Feeder Cells ; Genes, Tumor Suppressor ; Recombination, Genetic ; Ribonucleoproteins ; Telomerase* ; Telomere ; Up-Regulation