BACKGROUND: The investigation of culturing, passaging and establishing human limbal stem cells can strengthen the recognition of the stem cells and provide the enough cellular reserve for the basic and clinical research of limbal stem cell transplantation.OBJECTIVE: To explore a method of pessaging and establishing cell line of human limbal stem cells cultured in vitro.DESIGN: Randomized controlled observation.SETTING: Gannan Medical College.MATERIALS: The experiment was performed at Scientific Center of Gannan Medical College and the National Key Laboratory of Ophthalmology Hospital Affiliated to Sun Yat-Sen University from June 2003 to April 2004. Fresh human limbus corneae were isolated from two healthy donors. Procedures were performed according to the informed consent of the donors. Main reagents contained RPMI-1640 (Sigma R8755, containing L-glutamine) and 200 g/L fetal calf serum (FCS) (Gibco 16140-071). DMEM medium, chondroitin sulfatase and human epidermal growth factor (hEGF) were purchased from Sigma Co. USA; HEPES and DMSO were bought from Gibco, USA; 100% glycerinum was purchased from Yunjia Huangpu Pharmaceutical Product Limited Company, PRC; glutaraldehyde was bought from E.Merk, Germany; Alcohol, chlorhydric acid, acetone and methyl aldehyde were purchased from Beijing Chemical Agent Company, PRC; 0.25% parenzyme was bought from Shanghai Xinhua Pharmaceutical Factory, PRC.Above-mentioned reagents were analytical pure grade.METHODS: After digestion, human limbal tissues in limbal basilar part with an abundant pigment were cultured in the culture flask containing RPMI-1640 and 200 g/L FCS and in culture dish containing amniotic extracellular matrix (AECM) as the cultural supporter. Primary and passage cells were observed under light microscope and scanning electron microscope (SEM). The revival ratio of stem cell refrigeration of every generation was calculated by the trypanblau exclusion experiment.MAIN OUTCOME MEASURES: ① Observational results of limbal stem cells during the primary culture and serial subcultivation in vitro, and ② revival ratio of stem cell refrigeration.RESULTS: ①Findings of primary culture: Most limbal stem cells in the culture flask had the adherence and were arrayed uniformly sparsely to form monolayer and adhered to the bottom of culture flask under the inverted phase contrast microscope after 1-day culture. ② Findings of serial subcultivation: After human epidermal growth factor (hEGF) was added into the second passage, cells were scattered into the monolayer and adhered to grow quickly.Morphological variability of all the cells increased obviously when passage the 30th generation. The cellular volume was obviously increasing, and the round or irregular round cells gathered together. The 33rd generation human limbal stem cells still could vigorously differentiate, proliferate and grow in ACEM. ③ The revival ratio of stem cell refrigeration was 82.2%.CONCLUSION: The human limbal stem cell lines were preliminarily established by culturing and freezing the cells of 33 generations in vitro. The human limbal stem cell lines preferred to grow in the culture dish containing AECM as the cultural supporter.

Objective To compare the effects of mini-midline catheters that were placed in different sites.Methods The inpatients of a tertiary general hospital in Zhejiang Province from October 2023 to January 2024 were selected as the study subjects using a fixed point continuous convenience sampling method.The patients were divided into 2 groups by simple random grouping method.The experimental group had a mini-midline catheter placed in the upper arm,and the control group had a mini-midline catheter placed in the forearm.The incidence of catheter-related complications,the puncture success rate with one-attempt,the total procedure time,the time of the first occurrence of catheter-related complications,the rate of removal due to complications,and the indwelling catheter duration were compared between the 2 groups.Results A total of 121 patients were included,including 64 in the experimental group and 57 in the control group.The incidence rates of catheter-related complications in the experimental group and the control group were 29.69％and 66.67％;the times of the first occurrence of catheter-related complications were 167(122,220)h and 104(73,168)h;the rates of removal due to complications were 17.19％and 42.11％;the indwelling catheter duration was 171(124,258)h and 120(92,187)h;the differences between the 2 groups of these outcomes were statistically significant(P＜0.05).The puncture success rates with one-attempt in the experimental group and the control group were 96.88％and 96.49％;the total procedure times were 352(296,446)s and 370(295,430)s;the differences between the 2 groups of these outcomes were not statistically significant(P＞0.05).Conclusion Mini-midline catheters inserted in the upper arm can reduce the incidence of catheter complications and the rate of removal due to complications,prolong the time of the first occurrence of catheter-related complications and the indwelling catheter duration.