Objective:To establish Crx-iCreERT2 fluorescent reporter human embryonic stem cell lines using CRISPR/Cas9 technology and 3D retinal organoid culture.Methods:The target site sequence of H9 cell line was verified by polymerase chain reaction (PCR). SgRNAs were designed by CRISPR/Cas9 technique and their activity was detected.The most optimal sgRNA was selected according to the factors such as activity and specificity.After identification of the target vectors by restriction enzyme and sequencing, the target vectors were transferred to the H9 cell line by electroporation.P2A-tdTomato-P2A-iCreERT2 was inserted between Exon4 and 3’-untranslated region of hES-ZLM-001 gene.Knockin positive clones were obtained after drug treatment, enrichment of positive clones.Primers were designed to perform PCR on the target region, and homozygous de-resistant knockin positive cell clones were selected according to the sequencing results and peaks.The 1-A07 cell line was cultured, and then flow cytometry for the proportion of OCT4 positive cells, immunofluorescence for three stem cell molecular markers including SOX2, NANOG, SSEA4, karyotype analysis were carried out to confirm whether the 1-A07 cell line could be used for further experiments.Retinal organoids were obtained by three-dimensional (3D) culture technology and the expression of molecular markers was detected by immunofluorescence at different developmental stages of retinal organoids. Results:The target site sequence of H9 cell line was consistent with that given by Genebank and Ensembl.Sixteen sgRNAs were designed according to the target site sequence of H9 cell line, and finally sgRNA8 and sgRNA12 were selected.The sgRNAs and recombinant plasmids were transfected into the H9 cell line by electroporation, and four homozygous de-resistant knockin positive cell clones were obtained by PCR.Crx-iCreERT2 fluorescent reporter human embryonic stem cell lines were successfully obtained.In 1-A07 cell line, the proportion of OCT4 positive cells was about 98.7% by flow cytometry, and the expression of three stem cell markers was positive by immunofluorescence, and the karyotype was normal 46, XX.The results showed that the 1-A07 cell line could be used for further experiments.The Crx-iCreERT2 fluorescent reporter human embryonic stem cell lines were differentiated into tdTomato positive retinal organoids by 3D culture technology.BRN3A positive ganglion cells, CALBINDIN positive horizontal cells and CHAT positive amacrine cells appeared on day 30 of differentiation.RECOVERIN positive photoreceptors arose on day 45 of differentiation.PKCα positive bipolar cells presented on day 90 of differentiation.Ganglion cells were shown in the deep layer of retinal organoids, and horizontal cells, amacrine cells and bipolar cells in the middle layer, and photoreceptors in the top layer.Conclusions:Crx-iCreERT2 fluorescent reporter human embryonic stem cell lines are successfully established and can be differentiated into retinal organoids that express tdTomato red fluorescence through 3D culture technology.Those retinal organoids contain the same types of neurons as normal human retinas, and follow a certain temporal and spatial developmental sequence similar to the developmental rules of normal human retinas.Crx-iCreERT2 fluorescent reporter human embryonic stem cell line is a powerful tool for researching retinal development and diseases and can be applied in treatments for blindness.

OBJECTIVE:To guarantee the reliability of cold chain drug storage device and the quality of the drugs in the hospi-tal. METHODS:The establishment and effects of drug cold chain monitoring platform in our hospital were introduced as well as re-lated improvement and measures. RESULTS:The cold chain monitoring platform was established in our hospital through equipping with related equipment, constructing platform network, determining monitoring point, setting early warning condition, etc. All-weather and real-time monitoring of temperature and humidity were realized for different districts of the hospital and different cold chain equipments. The platform could provide the information about operating condition of equipment,and complete drug re-frigeration temperature record;equipment failure could be dissolved in time based on early warning message. Through analyzing rea-sons of early warning message,the unqualified equipment was updated in time,which further improve the system and procedure of cold chain monitoring platform. Within 2 years after the implementation of the platform,53 sets of special refrigerator for drug were updated. Monthly early warning message and the rate of refrigeration equipment early warning decreased from 6869 and 100% in Sept. 2014 at the early stage of short message warning function to 1083 and 40% in Feb. 2016,respectively. CONCLU-SIONS:The establishment of drug cold chain monitoring platform guarantees the reliability of cold drug storage condition so as to ensure the quality of drug in the hospital.