Optimization of expression yield in a stable cell line expressing a novel mutated chimeric tissue plasminogen activator (mt-PA)

Abstract The development of stable cell lines producing recombinant proteins is very time-consuming and laborious. One of the practical approaches successfully performed is Fluorescence-Activated Cell Sorting (FACS). A mutated chimeric tissue plasminogen activator (mt-PA) was developed by removing the first three domains of t-PA, insertion of GHRP sequence and mutation toward resistance to plasminogen activator inhibitor-1 (PAI-1). In the current study, a new stable CHO-DG44 cell line producing mt-PA was developed by two sequential clonal selections: FACS and clonal-selection by limiting dilution. Furthermore, the expression was more evaluated using two different expression media. Finally, the high-producing clones were selected based on the dot blot and amidolytic activity test. The transfection efficiency of CHO-DG44 cells was 38% as measured by flow cytometry on green fluorescent protein (GFP). After performing FACS on stable cell pools, the expression yield was increased to fifty-fold. In terms of growth profile, CD-DG44 showed higher viability and cell density results than ProCHO5 medium. The expression of mt-PA was significantly higher in CD-DG44 than in ProCHO5, 765 and 280 IU/mL, respectively. Our data indicated that selection of an appropriate expression medium played a critical role in the development of potent producing stable cells by FACS.

Advances of high-throughput screening system in reengineering of biological entities / 生物工程学报

Enzymes and cell factories are the core of industrial biotechnology. They play important roles in various fields such as medicine, chemical industry, food, agriculture, and energy. Usually, natural enzymes and cells need to be engineered to improve the catalytic efficiency, stability and enantioselectivity. Directed evolution makes it possible to rapidly improve the properties of enzymes and cell factories. Sensitive and reliable high-throughput screening approaches are the key for successful and efficient engineering of enzymes and cell factories. In this review, we first summarize the advantages and disadvantages of different screening methods and signal generation strategies as well as their application scope; we then describe the latest advances of ultra-high throughput screening technology applied in the directed evolution of enzymes and cell factories in the past three years. On this basis, we discuss the limiting factors that need to be further improved for high-throughput screening systems and forecast the future development trends of high-throughput screening methods, hoping that researchers in various fields including biotechnology and instrument development can cooperate closely to enhance the reliability and applicability of the high-throughput screening techniques.

Epidural Fat-Derived Mesenchymal Stem Cell: First Report of Epidural Fat-Derived Mesenchymal Stem Cell

STUDY DESIGN: Experimental study. PURPOSE: To determine whether epidural fat (EF) tissue contains mesenchymal stem cells (MSC). OVERVIEW OF LITERATURE: Spine surgeons are unaware of the contents of EF tissue and the reason for its presence between the ligamentum flavum and the dura mater; therefore, EF tissues are routinely eliminated during surgical procedures. However, EF removal causes certain postoperative problems, such as post-laminectomy syndrome. We hypothesized that the EF tissue may play a significant supportive role for the neural structures and other nearby conditions. METHODS: EF tissues were obtained from consenting patients (n=3) during posterior decompression surgery of the lumbar spine. The primary cells were isolated and cultured as per previously described methods with some modifications, and the cell morphology and cumulation were examined. Thereafter, reverse transcription–polymerase chain reaction (RT-PCR), a fluorescence-activated cell sorting (FACS) analysis, and differentiation potency for differentiation into osteoblasts, chondroblasts, and adipocytes were investigated to identify whether the cells derived from EF are MSC. RESULTS: The cells from the EF tissue had a fibroblast or neuron-like morphology that persisted until the senescence at p18. MSC-specific genes, such as OCT4, SOX2, KLF4, MYC, and GAPDH were expressed in the RT-PCR study, while MSC-specific surface markers such as CD105, CD90, and CD73 were exhibited in the FACS analysis. The differentiation properties of EF-MSC for differentiation into the three types of cells (osteoblast, chondroblast, and adipocyte) were also confirmed. CONCLUSIONS: Based on the cell culture, FACS analysis, RT-PCR analysis, and differentiation potent outcomes, all the features of the cells corresponded to MSC. This is the first study to identify EF-MSC derived from the EF tissue.

Immortalization of spleen fibroblastic reticular cells infected by severe fever thrombocytopenia syndrome virus / 中华实验和临床病毒学杂志

Objective@#To isolate, purify and culture fibroblastic reticular cells (FRCs) of mouse in spleen, to develop a reliable and robust method to immortalize primary mouse FRCs, to filter stable FRCs cell lines, to prove that the clones can be infected by SFTSV in vitro.@*Methods@#After purifying FRCs by fluorescence activated cell sorting (FACS) from autoMACS-enriched stroma cells of mouse spleen, we infected FRCs by simian virus 40 large T antigen in vitro, screened the FRCs clones with puromycin, compared primary and immortalized FRCs by RNA sequencing(RNA-seq) technology, infected the clones with severe fever with thrombocytopenia syndrome virus (SFTSV) in vitro.@*Results@#We succeed in culturing purified FRCs from spleen, isolated four stable FRCs clones, two of which have a purity of 99%, survived for more than 50 passages, express the key FRCs marker podoplanin and do not express CD31 and CD45. Clone 01 lost the typical FRCs-like morphology, the rate of expansion of which is quite different from that of primary FRCs and Clone 02. Clone 02 can be infected with SFTSV, which has the same gene expression pattern and immunophenotype with primary FRCs.@*Conclusions@#The stable FRCs clone Clone 02 has FRCs-like morphology and express key FRCs surface markers podoplanin (GP38 or PDPN) and do not express endothelial cell markers CD31 and leukocyte common antigen CD45. The RNA expression profiles identified by RNA-seq are also characteristic of FRCs. Infected with SFTSV in vitro, Clone 02 will be a new platform to study SFTSV.

Application of fluorescent-activated cell sorting (FACS) technique in the research of rabbit limbal stem cells / 眼科研究

Objective The fluorescence-activated cell sorting (FACS) technique is a method for the identification and isolation of different cell populations.At present,the special surface marker for limbal stem cells has been not found yet.This study aimed to investigate the application of FACS technique in the research of rabbit limbal stem cells.MethodsCorneal limbal tissue was obtained from New Zealand white rabbits and cultured using the explant culture method in SHEM.Side population cells (SP cells) and non-SP cells were sorted from cultured rabbit limbal epithelium cells by FACS at a excitation wavelength 350 nm,and acquistion length 450 nm (blue light) and 675 nm (red light).The SP cells and non-SP cells were identified by detecting the expression of ABCG2 and K3/K12.The colony-forming efficiency of SP cells and non-SP cells were evaluated by the observation of cellular vitality with trypan blue staining.The percentage of colony formation was calculated as the colony number in various group/200×100%.ResultsIn 48-72 hours after primary culture,limbal epithelial cells migrated from the cultured tissue mass to form the mambrane-like structure and achieved 70%-80% confluence.The cells showed round,polygon and flattened shape.The proportion of SP cells in cultured limbal epithelial cells was 0.22%±0.09% with a colony-forming efficiency of 5.52±0.45% in SP cells and 0.78%±0.73% in non-SP cells,with a statistically significant difference between the two populations (t＝2.17,P＜0.01).After verapamil,an inhibitor of the expression of the ABCG2 protein,was added into the medium,the proportion of SP cells in the cultured limbal epithelial cells declined to 0.04%±0.006%.The SP cells presented a positive immunoresponse for ABCG2 and absence of immunoresponse for K3/K12,but a contradictory staining result was found in non-SP cells.ConclusionFACS can be applied in the research of limbal stem cells.

Establishment of B-1 cell-derived polyreactive monoclonal antibodies and expression of costimulators by B-cell to antigenic stimulation / 대한치주과학회지

No abstract available.