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Adenoviridae , Adhesives , Cell Culture Techniques , Culture Media, Serum-Free , Fluorescence , Vero CellsABSTRACT
BACKGROUND: Corneal epithelial stem cells, also known as limbal stem cells, are distributed in the basal layer of limbal epithelium. It is extremely difficult to deal with limbal stem cell deficiency or dysfunction that is caused by severe thermal burn, chemical burn, and chronic inflammation of ocular surface. At present, in vitro culture of corneal epithelial stem cells using tissue engineering technology followed by clinical transplantation is a new and effective therapeutic direction. OBJECTIVE: To explore the feasibility of serum-free culture of human corneal epithelial stem cells in vitro using modified explant culture method. METHODS: The remaining donor corneal tissues after keratoplasty (less than 8 mm in diameter) were obtained from Henan Eye Bank, and the outer and middle limbus were dissected under surgical microscope. Two culture methods were used to culture human corneal epithelial stem cells. In the conventional explant culture group, the limbal tissues were adhered to the dish with the epithelium being upward, then Keratinocyte-serum free medium (K-SFM) was added into dishes, followed by incubation at 37 °C in a 5% CO2 incubator. In the modified explant culture group, limbal tissues were dissected to immerse in the K-SFM culture medium and incubated at 37 °C in the 5% CO2 incubator for 12 hours. The limbal tissues were then adhered to the dish with the epithelium being downward. The day whenever the cells from the limbal tissues adhered to the dish was marked as the 1st day of culture, and changes in cell morphology and growth were recorded by phase contrast microscopy every day. Immunofluorescent staining was used to detect the expression of K3 and p63 in primary cells on the 5th, 10th and 14th day of the modified explant culture. RESULTS AND CONCLUSION: The mean early stage of growth in the modified explant culture group was shorter than that of the conventional explant culture group (P < 0.05), and the mean growth rate of the modified explant culture group was higher than that of the conventional explant culture group (P < 0.05). In the modified explant culture group, cells had a good growth state, and many cells with small size gathered together on the 10th day of culture. On the 14th day, cell clones were formed, and the cells in the clone showed uniform morphology. On the 5th day, K3 highly expressed, while p63 lowly expressed in primary cells. On the 10th day, both of K3 and p63 had an increased expression. On the 14th day, there was no significant increase in the K3 expression, but the expression of p63 increased significantly. In the in vitro serum-free culture condition, the modified explant culture could significantly promote the growth of corneal epithelial stem cells, and expand corneal epithelial stem cells in vitro, which could provide sufficient seed cells for enriching corneal epithelial stem cells and constructing human limbal multilayered epithelial sheets.
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Objective:To isolate cell spheres containing cancer stem cells (CSCs) from lung cancer cell lines (A549 and H1299) and iden-tify their biological characteristics. Methods:By adopting the method of serum-free suspension culture, A549 cells and H1299 cells were cultured on no-adhesion plate to form tumor spheres. Clone assay, CCK-8 assay, and Transwell assay were employed to observe proliferation, self-renewal and invasion of tumor spheres. Besides, RT-PCR was performed to compare the expression levels of stem cell markers between sphere cells and adherent cells. Adherent A549 cells and A549 cell spheres were inoculated subcutaneously in nude mice and the tumor growth was assessed. Results:Isolated CSCs from A549 cells and H1299 cells in serum-free medium (SFM) without adhesion could grow as floating cell spheres. The results demonstrated that the self-renewal, proliferation and invasion of A549 and H1299 sphere cells were stronger than parent cells (P<0.05). When compared with adherent cells, the mRNA level of expres-sion of cell spheres' stem cell markers (Oct4 and Nanog) were significantly high (P<0.05) and A549 spheres had a stronger tumorigenici-ty in nude mice. Conclusion:SFM without adhesion can be a quick and easy method to construct stem cells model from human lung adenocarcinoma cell lines A549 and H1299.
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Objective To generate suspended spheres ,Human colon cancer cell line SW620 cells were acclimated gradually in the free serum culture medium ,1 week later ,a great quantity of spheres were obtained for the further research .Methods SW620 cells and spheres were incubated with the following Abs :anti‐SSEA‐4 Ab and anti‐TRA‐1‐60 Ab and detected with confocal microscopy . SW620 cells and spheres were collected and stained by PI ,Cell cycle distribution was then acquired by flow cytometry .SW620 cells and spheres were transferred into Adipocyte Differentiation Medium and cultured for 7 d and then lipid droplets were counted . SW620 cells and sphere cells were injected subcutaneously into the lateral root of one posterior limb of a nude mouse ,tumor volume was calculated .Results Spheres formed when grown under serum‐free conditions in 7 days ,and the ability of SW620 cells to form spheres was sustained .A few of SW620 adherent cells be cultured in DF+10% FBS clearly showed the expression of pluripotent stem cell markers Ssea‐4(25 .739 ± 7 .62)% ,Tra‐1‐60(27 .742 ± 4 .311)% ,but almost all spheres expressed the 2 markers and there was a significant difference between the two groups in the expression ratio (P<0 .05) .It had been shown that the spheres exhibited a relatively high proportion of cells in G0/G1 phase(84 .19 ± 2 .52)% and low proportion in S phase(7 .18 ± 1 .35)% ;when com‐pared to SW620 cells ,G0/G1 phase(63 .02 ± 6 .73)% ,S phase(20 .89 ± 3 .84)% (P<0 .05) .Adipocytes drops from spheres and ad‐herent cells were calculated under microscope after been cultured in adipocytes liquid for 7 d .The number of adipocytes drops in spheres was (583 .80 ± 77 .69) ,but(169 .20 ± 26 .43)in SW620 adherent(P<0 .05) .2 × 104 sphere cells injected subcutaneously into the lateral root of one posterior limb of a nude mouse fromed transplantation tumor after 4 weeks ,and the tumor size was (2 279 .98 ± 346 .27) mm3 ;2 × 105 adherent cells injected subcutaneously into the lateral root of one posterior limb of a nude mouse fromed transplantation tumor after 4 weeks ,and the tumor size was(889 .75 ± 78 .79) mm3 ,(P<0 .05) .Conclusion In this study , it is successfully observed that SW 620 cells being cultured in serum‐free medium resulted in spheres rapidly and the spheres are se‐rially passaged with relatively high efficiency .The spheres show the characteristics of the cancer stem cell .
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[ ABSTRACT] AIM:To cultivate stem-like spheres from SW620 cell line in the specific serum-free medium and evaluate the features of the cancer stem cells, and to investigate the effects of docosahexaenoic acid ( DHA) and eicosapen-taenoic acid ( EPA) on the growth of SW620 stem cell-like cells.METHODS: Human colon cancer stem cell-like cells ( CSCLC) were obtained from SW620 spheres cultured in serum-free medium.These cells were tested for the expression of SSEA-1 and TRA-1-81 by immunofluorescence staining.The mRNA expression of Sox-2 and Oct-4 was detected by real-time PCR.The efficiency of colony formation on a soft agar gel and tumor formation in the nude mice was compared between SW620 adherent cells and CSCLC.The inhibitory effects of 5-fluorouracil (5-FU) and mitomycin C on both types of cells were measured by MTS assay.MTS assay, Annexin V/PI staining and trypan blue staining were used to determine the effects of DHA and EPA on both types of cells.MTS assay was also used to analyze the combined effect of DHA or EPA with chemotherapeutic drugs on SW620 CSCLC.RESULTS:SW620 cells formed spheres in serum-free culture.The cells from spheres highly expressed SSEA-1 and TRA-1-81, transiently expressed Sox-2 and Oct-4 genes and were more resistant to 5-FU and mitomycin C treatments.These cells exhibited a greater ability in clone formation and tumorigenicity, indica-ting that these cells carried stem cell-like features, hence were considered SW620-derived CSCLC.DHA and/or EPA sup-pressed SW620 CSCLC by inhibiting cell growth, inducing cell apoptosis and sensitizing them to chemotherapeutic drugs. CONCLUSION:The cells with stem cell-like features, such as high efficiency in clonogenicity, tumorigenicity and resist-ance to chemotherapeutic drugs, can be obtained from SW620 spheres cultured in serum-free condition.DHA and EPA in-duce apoptosis in SW620-derived CSCLC and sensitize them to chemotherapeutic drugs.
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OBJECTIVE: This study was conducted to examine the influences of supplementation of the serum substituents and available period of serum-free Vero cell conditioned media (SF-VCM) manufactured from Dulbecco's modified Eagle medium cultured with Vero cells for in vitro development of mouse preimplantation embryos. METHODS: A total of 1,099 two-cell embryos collected from imprinting control region mice were cultured in SF-VCM with 10% and 20% human follicular fluid (hFF), serum substitute supplement (SSS), and serum protein substitute (SPS). Development of embryos was observed every 24 hours. Results between different groups were analyzed by chi-square test, and considered statistically significant when P-value was less than 0.05. RESULTS: The rates of embryonic development cultured in SF-VCM supplemented with serum substituents were significantly higher compare with serum-free group (P < 0.05). The rates of embryonic development after 48 hours (morula< or =) and 96 hours (blastocyst< or =) were significantly higher in 20% SSS and 10% SPS than in 20% hFF supplementation (P < 0.05). And the rates of embryonic development after 96 hours (hatching blastocyst< or =) were significantly higher in 10% SPS (94.5%) than in 20% SSS (82.6%) and 20% hFF supplementation (68.5%). The rates of embryonic development according to storage period of the SF-VCM supplemented with 10% SPS showed no significant difference between control, 2 weeks and 4 weeks group. However developmental rate in 6 weeks storage group was significantly lower than other groups. CONCLUSION: The rate of embryonic development after 96 hours (hatching blastocyst< or =) was significantly higher in SF-VCM supplemented with 10% SPS. And storage period of media up to 4 weeks did not affect on embryonic development.
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Animals , Female , Humans , Mice , Pregnancy , Coculture Techniques , Culture Media, Conditioned , Eagles , Embryonic Development , Embryonic Structures , Follicular Fluid , Vero CellsABSTRACT
Human fibroblasts that maintain the structural integrity of connective tissues by secreting precursors of the extracellular matrix are typically cultured with serum. However, there are potential disadvantages of the use of serum including unnatural interactions between the cells and the potential for exposure to animal pathogens. To prevent the possible influence of serum on fibroblast cultures, we devised a serum-free growth method and present in vitro data that demonstrate its suitability for growing porcine fetal fibroblasts. These cells were grown under four different culture conditions: no serum (negative control), 10% fetal bovine serum (FBS, positive control), 10% knockout serum replacement (KSR) and 20% KSR in the medium. The proliferation rates and viabilities of the cells were investigated by counting the number of cells and trypan blue staining, respectively. The 10% FBS group showed the largest increase in the total number of cells (1.09 x 10(5) cells/ml). In terms of the rate of viable cells, the results from the KSR supplementation groups (20% KSR: 64.7%; 10% KSR: 80.6%) were similar to those from the 10% FBS group (68.5%). Moreover, supplementation with either 10% (3.0 x 10(4) cells/ml) or 20% KSR (4.8 x 10(4) cells/ml) produced similar cell growth rates. In conclusion, although KSR supplementation produces a lower cell proliferation rate than FBS, this growth condition is more effective for obtaining an appropriate number of viable porcine fetal fibroblasts in culture. Using KSR in fibroblast culture medium is thus a viable alternative to FBS.
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Animals , Humans , Cell Proliferation , Connective Tissue , Diminazene , Extracellular Matrix , Fibroblasts , Trypan BlueABSTRACT
The cancer stem cells(CSCs)from human osteosarcoma by serum-free three-dimensional culture combined with anticancer drugs were isolated and identified.The primary cells derived from human osteosarcoma were digested by trypsin to prepare a single-cell suspension,and mixed homogeneously into 1.2% alginate gel.Single-cell alginate gel was cultured with serum-free DMEM/F12 medium.Epirubicin(0.8 μg/mL)was added to the medium to enrich CSCs.After cultured conventionally for 7 to 10 days,most of cells suspended in alginate gel were killed by epirubicin.But few cells survived and some single-cell cloning spheres formed.Immunofluorescent staining for Oct3/4 and Nanog was implemented to find cells with properties of self-renewal and multi-potential differentiation.Cells from cloning spheres were transplanted into BALB/c mice to detect the tumorigenicity in vivo.The results showed that some cells positive for Oct3/4(TRITC)and Nanog(TRITC)were found in single-cell cloning spheres,and most of positive cells were concentrated in the core of sphere.Cells from spheres could form osteosarcoma in the body of mice.It was concluded that cells from single-cell cloning spheres had the properties of the expression of parts of stem cell genes(Oct3/4 and Nanog),resisting anti-cancer drugs,and tumorigenicity in vivo.To sum up,it is believed that cells obtained from osteosarcoma by serum-free three-dimensional culture combined with anticancer drugs are cancer stem cells.
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Objective:To investigate whether CD3McAb activated killer(CD3AK)cells could induce apoptosis in HL 60 cells in serum free culture system.Methods:HL 60 cells were coincubated with CD3AK cells expanded in serum free medium,then apoptosis was assessed by means of morphological studies,DNA agarose gel electrophoresis and flow cytomether(FCM) analysis.Results:After 7 h of coincubation with CD3AK cells in serum free medium,HL 60 cells showed morphological features of apoptosis and typical DNA ladder on gel electrophoresis.FCM analysis showed that 24.59% cells were found to undergo apoptosis.Conclusion:CD3AK cells can induce apoptosis in HL 60 cells under serum free culture conditions.
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During the past few years much effort has been put into simplifying the clinical man-agement of in-vitro fertilization/embryo transfer cycles. One important step was the intro-duction of transvaginal ultrasound-guided oocyte collection, as previously described. This study describes further simplifications in the clinical management of ovarian stimulation and luteal support, and in-vitro fertilization procedure. During the period from October 1994 to September 1995, two major simplification steps were introduced. All cycles were administe-red with a gonadotrophin-releasing hormone agonist according to a long or short protocol preventing premature LH surge. During period I (Group I, n=62 cycles), closer monitoring by several pelvic ultrasound scans and serum oestradiol was used for monitoring the ovarian stimulation ; HTF media with fetal cord serum was used for insemination, growth and tran-sfer media in IVF-ET procedure ; progesterine in oil was daily used by intramuscular injec-tion for luteal support. During period II(Group II-I, n=71 cycles), only several ultrasound scans were used for monitoring the ovarian cycle ; Medi-cult IVF media containing synthetic serum replacement was used for insemination, growth and transfer media; Progesterine in oil was used daily by intramuscular injection for luteal support. During period III(Group II-II, n=16 cycles), further simplification of the clinical management was introduced by using a intravaginal micronized progesterone(Utrogestan) for luteal support. Retrospective analysis between Group I and Group II showed no differences in the number of oocyte(13.2+/-0.8/14.6+/-1.0), fertilization rate(71.5 %/60.7 %), cleavage rate(63.6 %/57.9 %), number of embryos transfered(5.0+/-0.5/4.5+/-0.5). Ongoing pregnancy rates obta ined from the three groups(Group I, Group II-1, Group II-II) were 25.8 %, 25 % and 40 %, respectively(p=ns). But introduction of minimal monitoring gave a significant reduction in the average number of US measurements in the simplified groups(Group II) compared with the group using the conventional monitoring protocol(Group I)(3.8+/-1.0/8.7+/-2.8, p<0.05). In the above groups, five patients developed severe OHSS but there was no differenc e in the distribution. Conclusively, simplified protocols including minimal follicle monitoring only by US, IVF-ET with Medi-cult IVF media containing synthetic serum replacement and the luteal support with intravaginal micronized progesterone gave a increased efficacy of the clinical phase of IVF treatment without a reduction in the success rate.
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Female , Humans , Culture Media, Serum-Free , Embryo Transfer , Embryonic Structures , Estradiol , Fertilization , Injections, Intramuscular , Insemination , Menstrual Cycle , Oocyte Retrieval , Ovulation Induction , Pregnancy Rate , Progesterone , Retrospective Studies , UltrasonographyABSTRACT
The establishment of a serum-free culture medium (SFM) for SSV-NIH3T3 cells is reported in this paper. In this medium DMEM/Ham's F12 is the fundamental element in basal medium, insulin and undefatty bovine serum albumin are key elements among the supplements. In this SFM SSV-NIH3T3 cells grow well, they keep the ability of secreting platelet-derived growth factor-like material into culture medium and causing tumor growth in nude mice.