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Organoids are a novel disease model that is self-assembled from stem cells or malignant tumors and is used in clinical research. They are similar to tissues and organs in the body and have partially functional 3D cell structures. There are two types of traditional models for liver cancer research， i.e.，in vivo models （animal models of liver cancer established by induction） and in vitro cell experiments using corresponding cell lines. Organoids have the advantages of the two types of traditional models and show unique advantages in tumor research. Traditional models cannot fully reflect the microenvironment of cells， which often leads to the inconsistency with clinical research findings， and the emergence of new research models provides a new direction for the research on liver cancer. This article reviews the research advances in liver cancer organoids， in order to provide a new perspective for future research on liver cancer. [ABSTRACT FROM AUTHOR]

In vitro follicle culture has important implications for preserving fertility in patients with the decreased ovarian function caused by cancer treatment. It is also an effective method for studying follicle development and oocyte maturation. In order to establish an in vitro culture system that can better simulate the in vivo growth environment, current in vitro follicle culture systems are roughly divided into two -dimensional and three-dimensional cultures. Two-dimensional follicle culture systems have been successfully established in rodent models. However, since three-dimensional culture can provide the spatial structure for follicular growth, the effect of three-dimensional culture is superior to that of two-dimensional culture. Therefore, three-dimensional culture has been widely used for the culture of follicles in various animal models. Three-dimensional culture systems can be divided into scaffold-based and scaffold-free culture systems, and the choice of scaffold material has a direct impact on in vitro follicle development. Scaffold materials can be classified into natural and synthetic materials according to their source. Natural materials, such as alginate, collagen and fibronectin, have better biocompatibility than synthetic materials, but the adjustability of synthetic materials makes them promising. This review summarizes the effects of choosing a three-dimensional culture system on in vitro follicle development. [ABSTRACT FROM AUTHOR]

Objective To explore the differences between ceiling culture and insert culture for dedifferentiated fat (DFAT) cells in rats to provide research rationales for primary culture of DFAT cells. Methods Mature fat cells were isolated from rat inguinal adipose pad of eight 4-6-week-old Sprague-Dawley (SD) rats and DFAT cells harvested in vitro by dedifferentiation culture using ceiling and insert cultures. Cell morphology, time and amount of complete culture medium required from isolation to first passage were compared. Purity and function of differentiation into smooth muscle cells of DFAT cells were compared by flow cytometry analysis and cell immunofluorescence. Results DFAT cells obtained by two methods both were fibroblast-like, spindle-shaped and peak-valley-like. The time required for DFAT cells cultured by ceiling culture and insert culture to grow to the first passage were (16.00±1.41) and (16.25±0.96) days respectively and the difference was not significant (P>0.05). The surface antigen phenotypes of DFAT cells cultured by two methods were positive for CD29, CD90 and negative for CD31, CD45;flow cytometric results of DFAT cells cultured by ceiling culture showed CD29 was expressed in (97.63±3.15)% cells, CD90(98.63±0.83)%, CD31(0.11±0.12)%, CD45(0.18±0.11)%; the results of DFAT cells cultured by insert culture showed CD29 was expressed in (96.93±3.61)% cells, CD90 (98.65±0.84)%, CD31 (0.16±0.13)%, CD45 (0.16±0.10)%. And the differences were not significant (P>0.05). DFAT cells cultured by ceiling and insert cultures could differentiate into smooth muscle cells and express αSMA and SMMHC after induction in vitro. The rates of αSMA positive cells were (73.00±7.88)% and (73.56±8.44)% respectively and the difference was not significant (P>0.05). Ceiling culture for DFAT cell growth to the first passage required more complete medium than insert culture[93.75(90.00, 93.75) vs 48.50(48.50, 48.50) ml]and the difference was significant (P < 0.05). Conclusion No significant differences exist in time expense, cell morphology, purity or function of induction differentiation into smooth muscle cells in vitro of DFAT cells cultured by ceiling and insert culture. However, from the perspective of economic expense, insert culture offers some advantage. [ABSTRACT FROM AUTHOR]

Objective: To explore the clinical phenotype and genetic variant in a Chinese pedigree affected with Hunter syndrome and create immortalized cell lines for the affected pedigree members., Methods: A pedigree of six members who had visited Xi'an Children's Hospital in July 2022 was selected as the study subject. Clinical data was collected. Whole exome sequencing was carried out for the pedigree members. Candidate variant was verified by Sanger sequencing. In addition, peripheral B lymphocytes were transfected with Epstein-Barr virus to create immortalized cell lines, which were then subjected to enzyme activity analysis., Results: The patient, a five-year-and-seven-month-old boy, had exhibited stiff limbs and enlarged joints. He had developed hernia, scaphocephaly, and barrel chest from 3 months of age. His uncle also had stiff limbs, poor hearing, blindness, and right oblique inguinal hernia. Above features had resembled those of Hunter syndrome. Genetic testing revealed that both the child and his uncle had harbored an IDS (NM&#95;000202.8): c.823G>A (p.D275N) variant, which was unreported previously. Bioinformatic analysis indicated that the D275 to be a highly conserved site, and the D275N variant may affect the stability of the protein's spatial conformation, thereby decrease the catalytic activity of the enzyme. The successfully constructed immortalized lymphoblastoid cell lines for the child and his parents showed increased volume, irregular shape, burr structure and cluster growth. And the value of IDS activity of the patient's immortalized lymphoblastoid cells was below the limit of detection. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as likely pathogenic (PS3+PM2&#95;Supporting+PM5+PP1+PP3)., Conclusion: Above finding has enriched the phenotypic and mutational spectra of Hunter syndrome, and provided a basis for the genetic counseling for this pedigree. The creation of immortalized cell lines has offered a model for further investigation of the impact of variant on the function of IDS and development of targeted drugs.

The adoptive immunotherapy mediated by tumor-infiltrating lymphocytes (TILs) has shown definite efficacy against various solid tumors. However, the inefficiency of the conventional method based on in vitro expansion of TILs fails to achieve the cell count and high tumor-killing activity required for therapeutic purposes. This study investigated the effect of 3D tumor spheroids on the activation and expansion of TILs in vitro , aiming to provide a novel approach for the expansion of TILs. We procured TILs and primary tumor cells from surgical samples of lung cancer patients and then compared the impacts of lung cancer cell line NCI-H1975 and primary lung cancer cells cultured under 2D and 3D conditions on the activation, expansion, and anti-tumor activity of TILs. Furthermore, we added the programmed cell death protein 1 (PD-1) antibody into the co-culture of primary tumor cells and TILs within a 3D environment to assess the effects of the antibody on TILs. The results showed that compared with 2D cultured tumor cells, the 3D cultured H1975 cells significantly enhanced the expansion of TILs, increasing the proportion of CD3 + /CD8 + cells in TILs to 61.6%. The 3D primary tumor model also enhanced the proportion of CD3 + /CD8 + cells in TILs (45.5%, 54.4%), induced apoptosis of tumor epithelial cells and decreased the overall tumor cells survival rate (16.7%) after co-culture. PD-1 antibodies further improved the in vitro expansion capacity of TILs mediated by 3D tumor spheroids, resulting in the proportions of 50.9% and 57.0% for CD3 + /CD8 + cells and enhancing the antitumor activity significantly (reducing the overall tumor survival rate to 9.36%). In summary, the use of 3D tumor spheroids significantly promoted the expansion and improved the anti-tumor effect of TILs, and the use of the PD-1 antibody further promoted the expansion and tumor-killing effect of TILs.

Objective: To investigate whether Schisandrin B (Sch B) can attenuate the effect of PM2.5 on the proliferation of human trophoblast HTR-8/Svneo (HTR) cells. Methods: The in vitro cultured HTR cells were treated by PM2.5 of 10-1 000 μg/mL concentration, Sch B-alone and Sch B plus PM2.5. The proliferation of HTR cells was tested by MTS method. Results: PM2.5 of different concentrations from 10 to 1 000 μg/mL inhibited the proliferation of HTR cells (P＜0.01), and the inhibition rate of 150 μg/mL PM2.5 was 21.97%. Sch B of the concentration from 0.1 to 5 μmol/L attenuated the PM2.5-induced inhibition of HTR cells proliferation, with a dose-effect relationship among 0.1-2 μmol/L. Sch B of 2 μmol/L presented the highest protection rate (14.07%). Conclusions: Sch B could attenuate the PM2.5-induced inhibition of HTR cell proliferation, suggesting a potential protection of human trophoblast cells in vivo [ABSTRACT FROM AUTHOR]

Spermatogonial stem cells (SSCs) are located on the basement membrane of the testicular seminiferous tubules, which can not only renew themselves but also differentiate to produce sperm. The characteristics of SSCs are critical in assisted reproductive technologies. After years of research, the great progress has been made in in vitro proliferation and differentiation of SSCs from rodent SSCs to human SSCs, which depends on the continuous improvement of in vitro culture methods to a certain extent. At present, the in vitro culture of SSCs has developed from two-dimensional culture to three-dimensional culture, such as soft agar threedimensional culture method, methylcellulose three - dimensional culture method, nanofiber scaffold three - dimensional culture method, and decellularized biological scaffold three -dimensional culture method. The continuous improvement of SSCs in vitro three-dimensional culture method is of great significance for revealing the mechanism of male germ cell generation and treating male infertility. In order to explore the technical methods for improving the in vitro culture efficiency of SSCs and establish the long-term and stable cell lines of SSCs, the research progress of in vitro three-dimensional culture methods of SSCs were reviewed in this paper. [ABSTRACT FROM AUTHOR]

Objective To establish a rapid and high efficient method of primary culture of BCAFs by improving primary culture methods of common breast cancer-associated fibroblasts (BCAFs). Methods We took cancerous tissue samples from the patients diagnosed as invasive breast cancer. All samples were randomly divided into two groups: the modified group and the conventional group with same size and quality. The modified group was digested with a modified enzyme digestion method (typeⅡcollagenase+hyaluronidase digestion for 10 min). The conventional group was digested with conventional enzymatic digestion method (typeⅠcollagenase digestion for 8h). The total number of cells obtained, the activity of adherent cells after 48h culture and the purity of BCAFs between two groups were compared by cell counting, MTT and immunofluorescence. The characteristics of primary BCAFs were observed under inverted microscope. Results The amount of cells, the activity of adherent cells after 48h culture and cells purity obtained by the modified enzyme digestion method were all higher than those by the conventional enzyme digestion method (P < 0.001). The primary BCAFs obtained were spindle-shaped or spindle-shaped, with many cytoplasmic particles and irregular cell arrangement. Conclusion A rapid and high efficient primary culture method of BCAFs is established successfully, as the foundation for the later study of its function in breast cancer.

The incidence rate of liver cancer has been rising in recent years. Traditional cell line culture and human patient-derived tumor xenograft models, which are commonly used tools to simulate the occurrence of human liver cancer, have deepened the understanding of tumor occurrence, development, and drug resistance mechanisms. However, they cannot reflect the accurate state of cancer cells, the tumor microenvironment, or spatial structural characteristics. Recently, more in vitro-produced physiological liver organoids have been applied in the study of liver cancer. Liver organoid models have made breakthroughs in the occurrence and development mechanisms of liver cancer, personalized drug screening and biomarker identification, immunotherapy, and regenerative medicine applications. This paper mainly summarizes the progress and application of liver organoids processed in the study of liver cancer.

BACKGROUND: Platelet-rich fibrin (PRF) prepared by autologous blood has become the hotspot in regeneration and repair of soft tissues. OBJECTIVE: To compare the effect of two kinds of PRFs on the proliferation activity of human gingival fibroblasts (HGFs), so as to provide reference for the selection of oral soft tissue regeneration and repair materials in the clinic. METHODS: Healthy gingival flaps from eight patients were collected when mandibular impacted teeth were removed. HGFs were obtained using tissue explant method, and then identified. The venous blood of the corresponding patients was drawn and made into advanced PRF and PRF, respectively, and passage 5 HGFs were divided into advanced PRF, PRF and blank groups for culture. The cell proliferation activity was detected by cell counting-kit 8 assay at 1, 3, 5, and 7 days. RESULTS AND CONCLUSION: The HGFs were positive for Vimentin staining and negative for Cytokeratin staining. Two kinds of PRFs were prepared successfully. The advanced PRF exhibited the deeper color, more thick membrane and better flexibility compared with the general PRF. Cell counting-kit 8 assay results revealed that at 1 day after culture, there was no significant difference in the proliferation activity among groups (P > 0.05). At 3 days, the cell proliferation activity in the advanced PRF and PRF groups was significantly higher than that in the blank group (P < 0.05). At 5 and 7 days, the order of cell proliferation activity was as follows: advanced PRF group > PRF group > blank group (P < 0.01, P < 0.05). To conclude, both advanced PRF and PRF can increase the proliferation activity of HGFs, especially the former. [ABSTRACT FROM AUTHOR]

BACKGROUND: The stem cell conditioned medium contains a large number of cell secretory factors contributing to wound repair. Liposome encapsulation facilitates the sustained release of cytokines at the wound site. OBJECTIVE: To study the effect of liposomes prepared from the conditioned medium of human umbilical cord mesenchymal stem cells on the healing of skin wounds in rats. METHODS: Human umbilical cord mesenchymal stem cells were isolated and cultured by tissue explant method, and cytokine detection was then performed. Twenty Sprague-Dawley rats were provided by the Experimental Animal Center of Shanxi Medical University in China to make skin wound models of 1 cm in diameter. Membrane dispersion technique was used to prepare the conditioned medium liposomal vesicles used for skin wounds in the rats. The skin and surrounding tissues of the repair site were taken for hematoxylin-eosin staining. The histological scores were used to judge the repair of wounds. RESULTS AND CONCLUSION: (1) The human umbilical cord mesenchymal stem cells were positive for CD73 (99.7%), CD90 (95.7%), CD105 (99.4%), CD45 (1.1%) and CD34 (0.1%). The cells could be induced to differentiate into osteocytes and adipocytes. (2) The concentrations of vascular endothelial growth factor, interleukin-8, and monocyte chemotactic protein 1 in the conditioned medium were 558.60 ng/L, 5 236.48 ng/L, and 2 972.55 ng/L, respectively. (3) The average particle size of liposome in the conditioned medium prepared by membrane dispersion method was 262.3 nm. Conditional culture of human umbilical cord mesenchymal stem cells and its liposome could increase the hair follicles and glands at the site of rat skin defects, the epidermis was thickened, and the histological score was markedly increased. There was no difference between the conditioned medium and conditioned medium liposome groups at 2 days of culture (P=0.730), but significant differences existed among the other groups (P < 0.05). To conclude, the human umbilical cord mesenchymal stem cell conditioned medium and its liposomes can promote the repair of skin defects in rats. [ABSTRACT FROM AUTHOR]

BACKGROUND: Placental mesenchymal stem cells can provide ideal seed cells for cell therapy, but trypsin cold digestion has not been used for cell culture. Therefore, we explored this method and compared it with the tissue explant method to screen a more convenient and more successful culture method. OBJECTIVE: To observe the biological features of human placental mesenchymal stem cells isolated by trypsin cold digestion and tissue explant method. METHODS: We isolated, purified, and subcultured mesenchymal stem cells (n=6) from human placenta by using trypsin cold digestion method and tissue explant method. The first appearance time and primary culture period of placental mesenchymal stem cells isolated using two methods were recorded. The growth curves of passage 3 cells from two culture methods were generated. Flow cytometry was used to analyze the expression of surface markers of passage 3 cells isolated using the two culture methods, and to study the neural and adipogenic differentiation of the cells. RESULTS AND CONCLUSION: Placental mesenchymal stem cells could be obtained using both culture methods. Adherent cells in the trypsin cold digestion group appeared significantly earlier than those in the tissue explant group (P < 0.05), and the primary culture period was also significantly shorter in the trypsin cold digestion method (P < 0.05). The growth curve of passage 3 cells showed a higher cell number at plateau growth using trypsin cold digestion than tissue explant method (P < 0.05). The cells isolated using two culture methods were positive for CD29 and CD105, but negative for CD34 and CD45, indicating the same cell phenotypes. After the induction, placental mesenchymal stem cells cultured by both methods had the potential to differentiate into nerve cells and adipocytes. Therefore, both culture methods can produce placental mesenchymal stem cells, but trypsin cold digestion method is more suitable for the culture of this kind of cells as compared with the tissue explant method. [ABSTRACT FROM AUTHOR]

BACKGROUND: Neuron culture and cell transfection technologies are important means to study the development and pathophysiologic mechanism of neurons in vitro, but the purity and transfection efficiency of primary cultured cortical neurons are poor. OBJECTIVE: To establish a simple, efficient and stable method of culture and transfection method of primary neurons in vitro. METHODS: Primary cortical neurons were harvested from neonatal 1-day rat brains under aseptic condition, which was digested with 0.25% trypsin prior to centrifugation and made into cell suspensions, followed by being seeded into Neurobasal-A medium (5×109 L-1 per pore). The morphological characteristics of neurons were observed by inverted microscope; two neuron-specific markers (MAP2 and Tuj1) were used for immunolabeling to identify the cultured cells; the transfection efficiency of neurons was tested by Lipofectamine 2000 and Block-iT Transfection Kit. RESULTS AND CONCLUSION: The neurons cultured in vitro exhibited interconnected networks after culture for 7 days. All the cultured neurons displayed MAP2 and Tuj1 immunoreactivity. The highly effective transfection was observed under fluorescence microscope using Lipofectamine 2000 transfected neurons. In summary, the culture method of primary cerebral cortex neurons can be adopted to obtain highly purified neurons. Besides, high transfection efficiency of primary neurons can be realized by artificial liposome. [ABSTRACT FROM AUTHOR]

In response to the market demand for therapeutic antibodies, the upstream cell culture scale and expression titer of antibodies have been significantly improved, while the production efficiency of downstream purification process is relatively fall behind, and the downstream processing capacity has become a bottleneck limiting antibody production throughput. Using monoclonal antibody mab-X as experimental material, we optimized the caprylic acid (CA) precipitation process conditions of cell culture fluid and low pH virus inactivation pool, and studied two applications of using CA treatment to remove aggregates and to inactivate virus. Based on the lab scale study, we carried out a 500 L scale-up study, where CA was added to the low pH virus inactivation pool for precipitation, and the product quality and yield before and after precipitation were detected and compared. We found that CA precipitation significantly reduced HCP residuals and aggregates both before and after protein A affinity chromatography. In the aggregate spike study, CA precipitation removed about 15% of the aggregates. A virus reduction study showed complete clearance of a model retrovirus during CA precipitation of protein A purified antibody. In the scale-up study, the depth filtration harvesting, affinity chromatography, low pH virus inactivation, CA precipitation and depth filtration, and cation exchange chromatography successively carried out. The mixing time and stirring speed in the CA precipitation process significantly affected the CA precipitation effect. After CA precipitation, the HCP residue in the low pH virus inactivation solution decreased 895 times. After precipitation, the product purity and HCP residual meet the quality criteria of monoclonal antibodies. CA precipitation can reduce the chromatography step in the conventional purification process. In conclusion, CA precipitation in the downstream process can simplify the conventional purification process, fully meet the purification quality criterion of mab-X, and improve production efficiency and reduce production costs. The results of this study may promote the application of CA precipitation in the purification of monoclonal antibodies, and provide a reference for solving the bottleneck of the current purification process.

BACKGROUND: Methods for extracting primary macrophages from rats include bronchoalveolar lavage fluid (BALF) extraction, spleen extraction, and peripheral blood extraction. These methods have their own advantages and disadvantages, and the cells show different activities, status and quantities. OBJECTIVE: To analyze the better selection of primary extraction and culture of macrophages from three different tissue sources. METHODS: Under the same experimental conditions, primary rat macrophages were cultured using three different methods: BALF extraction, spleen extraction and peripheral blood extraction. RESULTS AND CONCLUSION: The average numbers of primary macrophage extracted from BALF, spleen and peripheral blood were (1.14±0.14)×106, (9.29±0.19)×106 and (3.17±0.13)×106 respectively, there was a significant difference among groups (P < 0.001). The average survival time of the three methods for cell extraction was (111.00±5.98), (100.00±4.60) and (97.00±3.43) hours respectively, the difference was significant (P < 0.001). The activity of cells extracted from BALF was significantly higher than that of the other two tissues (P < 0.05), but there was no significant difference between spleen and peripheral blood (P > 0.05). These results indicate that BALF extracts the most activated macrophage can be extracted in BALF, and the largest number of macrophages can be extracted from the spleen. [ABSTRACT FROM AUTHOR]

BACKGROUND: Serum concentrations in culture medium influence the proliferation and differentiation of stem cells. OBJECTIVE: To study the proliferation and adipogenic differentiation potential of adipose-derived stem cells under culture with different concentrations of fetal bovine serum. METHODS: The mouse adipose-derived stem cells were harvested from subcutaneous adipose tissue of C57 mice at 7-10 days of age. Concentrations of fetal bovine serum in the culture medium were set to 5%, 10%, 15% and 20%. The passage 3 cells were used for subsequent studies. The proliferation of cells cultured with different serum concentrations was tested using the cell counting kit-8 assay. The adipogenic differentiation potential of cells was evaluated with Bodipy staining. Quantitative RT-PCR and western blot assay were used to determine expression levels of Cebpa and Pparg mRNA as well as C/EBP-α and PPAR-γ protein levels. RESULTS AND CONCLUSION: There was no statistical difference in the cell proliferation among cells cultured with different serum concentrations, even though adipose-derived stem cells cultured with 20% fetal bovine serum showed lower absorbance value at 450 nm than those cultured with 5%, 10% and 15% fetal bovine serum. The expression levels of Cebpa and Pparg mRNA and C/EBP-α and PPAR-γ protein in the 5% and 10% groups were considerably higher than those of the 15% and 20% groups, and the expression levels were highest in the 10% group. To conclude, different concentrations of fetal bovine serum have no significant effects on the proliferation of adipose-derived stem cells, while the adipogenic differentiation potentials of cells are different when cultured with different serum concentrations. High serum concentration can inhibit the adipogenic differentiation of adipose-derived stem cells. [ABSTRACT FROM AUTHOR]

BACKGROUND: To obtain enough adipose-derived stem cells (ADSCs) is the premise of repairing osteoporotic bone defects by autograft transplantation; therefore, how to efficiently, simply and economically harvest primary autologous ADSCs is the key to the treatment of osteoporosis. OBJECTIVE: To isolate and culture ADSCs from mice with osteoporosis (OP-ADSCs) in vitro by tissue explant culture and collagenase digestion, and to compare the efficacies of these two methods. METHODS: Adipose tissues were isolated from the inguen of C57BL/6 mice, from which OP-ADSCs were obtained by tissue explant culture and collagenase digestion respectively. Then, the cells were subjected to primary culture and subculture. The surface specific antigens of passage 3 cells were observed using flow cytometry, while the yields and proliferation abilities of passage 3 were compared at 7 days of culture. The adipogenic and osteogenic differentiation of OP-ADSCs at passage 4 was detected. RESULTS AND CONCLUSION: (1) The expression levels of CD34, CD146, Sca-1 in passage 3 cells were (15.22±1.85)%, (75.55±3.36)% and (83.48±4.22)% for the tissue explant culture and (13.46±2.21)%, (76.62±2.47)% and (84.84±3.56)% for the collagenase digestion method, respectively. (2) The cell yield from each milligram of adipose tissue by tissue explant culture was significantly higher than that by collagenase digestion (P < 0. 05). (3) The proliferation rate of cells in the tissue explant culture group was higher than that in the collagenase digestion group after 24 hours of culture. (4) The OP-ADSCs cultured by the two methods could differentiate into adipocytes and osteoblasts, and the lipid accumulation and the mineralized nodules showed no significant difference between the two groups (both P > 0.05). These experimental findings indicate that the tissue explant culture is more suitable for obtaining OP-ADSCs in vitro as compared with collagenase digestion, which contributes to provide adequate cell sources for studies on ADSCs treatment of osteoporotic fractures and bone defects. [ABSTRACT FROM AUTHOR]

BACKGROUND: Studies on rhesus monkey bone marrow mesenchymal stem cells (BMSCs) are limited, and the differentiation potential of juvenile rhesus monkey BMSCs remains unknown. OBJECTIVE: To investigate the biological characteristics of rhesus monkey BMSCs isolated and cultured in vitro, and to identify the growth curve and phenotype of BMSCs. METHODS: BMSCs from juvenile rhesus monkeys were isolated and cultured by density gradient centrifugation and the whole bone marrow adherence methods. BMSCs at passages 2 and 7 were taken to draw cell growth curves by MTS method. Flow cytometry was used to detect apoptosis of passage 3 BMSCs and to detect expression of cell surface markers, CD29, CD34, CD45, CD90, CD147, in passage 4 BMSCs. Osteogenic and adipogenic abilities of passage 3 BMSCs were also detected. RESULTS AND CONCLUSION: The BMSCs at passages 2 and 7 were incubated at first 2 days, logarithmically grew at 3-7 days, and then proliferated slowly at 8 days. The BMSCs at passage 2 showed stronger proliferation than those at passage 7. At passage 3, BMSCs were negative for CD34 and CD45, but positive for CD90, CD29 and CD147. Results from the flow cytometry showed that there were 73.83% living cells, 21.76% dead cells, and 4.27% apoptotic cells. BMSCs could differentiate into osteoblasts and adipocytes under osteogenic and adipogenic induction, respectively. To conclude, BMSCs from the rhesus monkey have strong proliferation and multilineage differentiation abilities in vitro. [ABSTRACT FROM AUTHOR]

Objective To explore the collagenase digestion combined with tissue pieces culture method for isolating and purifying rabbit lung fibroblasts. Methods Firstly，newborn rabbit lung were cut into tissue pieces. After the tissue pieces were digested mildly with collagenase type Ⅳ，they were cultured adherently，then lung fibroblasts were separated and purified. Thus original generation culture and subculture were carried out，and the cell growth curve was determined by using the MTT method. Results When tissue pieces were cultured adherently for 2 d，a small amount of lung fibroblasts climbed out from surrounding of tissue pieces. A large number of lung fibroblasts climbed out on 4 d，grew rapidly and were close to fusion on 7 d，which reached high fusion on 9 d. Parenchymal cells were basically wiped out after twice passages. The 1-4 generations had good cell activity status，and the cell proliferation ability was declined gradually after 5-6 generations. The cells appeared the gradual aging phenomenon. Conclusion The collagenase type Ⅳ mild digestion combined with tissue pieces adherent culture is a reliable and fast cultivation method of lung fibroblasts isolation and purification，which can obtained lung fibroblasts with typical morphology characteristics and good activity. [ABSTRACT FROM AUTHOR]

BACKGROUND: There are a lot of studies on isolation and culture methods of human placental chorionic-derived mesenchymal stem cells (hpcMSCs), but how to simply and efficiently harvest a large amount of primary MSCs has not been resolved. OBJECTIVE: To optimize the tissue explants method of isolating and culturing hpcMSCs in vitro. METHODS: Human placental chorionic villi were collected from full-term deliveries under aseptic condition and isolated by electric homogenizer. hpcMSCs were prepared by tissue explants method. The fluid and tissue of the primary culture flask and douching normal saline of the initial culture were centrifuged and prepared for secondary culture. RESULTS AND CONCLUSION: It saved time and effort to treat human placental chorionic villi with electric homogenizer, with good effects on tissue dispersion and removal of red blood cells. The average time of cell acquisition in initial culture and secondary culture was (17.73±1.14) and (10.03±1.30) days, respectively. The yields of primary cultured cells in initial culture and secondary culture were (6.97±0.98)×105 and (13.82±1.44)×105 per Φ100 mm culture dish, respectively. The adherent cells showed fibroblast-cell-like shape, which were in parallel or circinate arrangement. Highly expressed CD73, CD105 and CD90 could be detected in the third generation of hpcMSCs, but CD34, CD45, CD14, CD19 and HLA-DR were negative. Following induction, alizarin red staining and oil red O staining produced a strong reaction in cells. In a word, the optimized method is a simple and efficient method for obtaining a large amount of primary hpcMSCs. [ABSTRACT FROM AUTHOR]

BACKGROUND: As current studies on isolation, culture and cryopreservation of human amniotic epithelial cells (hAECs) are relatively scattered, it is difficult to form a comprehensive and effective solution to meet the clinical needs of stem cells for transplantation in future. OBJECTIVE: To establish the technology of isolation, culture and cryopreservation of hAECs, and to study the biological characteristics of hAECs. METHODS: Orthogonal method was used to study the effects of different factors on the separation, culture and cryopreservation, and range method was adopted to analyze the data to optimize the separation, culture and cryopreservation. We performed cell primary and passage cultures, morphology observed by microscope, drawn cell growth curve and flow cytometry assay, immunofluorescence staining, hepatocyte like cell differentiation to study the biological characteristics of hAECs. RESULTS AND CONCLUSION: (1) The optimal hAECs separation conditions were as follows: trypsin digestions were conducted at a concentration of 0.25%, four times, once for 20 minutes digestion; optimal conditions of culture were 4×108/L cell seeding density, 10 μg/L epidermal growth factor, 5% serum; optimal conditions of cryopreservation were 1×1010/L cell cryopreservation density, 10% dimethyl sulfoxide, 80% serum. (2) The primary cells were adhered to the wall in 2-3 days, exhibiting irregular polygon, paving stone-like growth. Cell adherence and growth rate were accelerated after subculture, and the growth and proliferation ability of passage 2 cells were not significantly decreased after cryopreservation and resuscitation. (3) Immunofluorescence staining showed that the primary cells strongly expressed SSEA-4 and CK19, but did not express Vimentin, CD45 and HLA-DR. The immunophenotype statistics of the primary and passage 4 cells showed the epithelial mesenchymal transition of hAECs in culture process. (4) Immunofluorescence staining showed that the liver cell marker expression of ALB, CK18 was significantly increased after hAECs were induced to differentiate into hepatocyte-like cells. Glycogen staining revealed glycogen synthesis in hAECs after 3 weeks of induction. To conclude, hAECs are easy to obtain and have strong proliferation ability in vitro, and express surface markers for undifferentiated embryonic stem cells. [ABSTRACT FROM AUTHOR]

BACKGROUND: Osteoblasts with high purity and activity are essential for bone metabolism research. OBJECTIVE: To explore a simple and effective culturing method of primary osteoblasts. METHODS: Osteoblasts were isolated from the parietal and frontal bones of newborn Sprague-Dawley rats using trypsin and collagenase digestion and tissue explant method. The morphology of osteoblasts was observed by inverted phase contrast microscope and transmission electron microscope; the cells was counted to draw the growth curve; the osteoblasts were identified by alkaline phosphatase BCIP/NBT staining and alizarin red staining. RESULTS AND CONCLUSION: The cells showed spindle, triangle or polygon shapes, having two or three protrusions. There were abundant mitochondria and endoplasmic reticulum under electron microscope, which presented the typical characteristics of osteoblasts. The cell growth was slow intially, accelerating at the 3rd day, and peaking at the 7th day. The cells were highly positive for alkaline phosphatase staining and were stained orangered through the alizarin red staining. To conclude, the cells isolated using enzymatic digestion combined with tissue explant method exhibit the typical characteristics and functions of osteoblasts, and this method is an ideal way to culture primary osteoblasts. [ABSTRACT FROM AUTHOR]

BACKGROUND: Currently, fibroblast-like synoviocytes from the synovial tissue of patients with rheumatoid arthritis or adjuvant arthritis rats are isolated and cultured mainly using enzyme digestion method, but tissue explant adherent method is rarely reported in primary culture. OBJECTIVE: To establish a culture method of fibroblast-like synoviocytes from adjuvant arthritis rabbits and to investigate cell biological characteristics. METHODS: Rabbit models of adjuvant arthritis were prepared by injected with adjuvant at multiple sites and times, the knee synovial tissues were obtained, and fibroblast-like synoviocytes were separated and cultured by the tissue explant adherent method. Furthermore, the cell morphology was observed. The cell proliferation was measured using cell counting kit-8 assay, and the expression of Vimentin was detected by immunofluorescence. RESULTS AND CONCLUSION: The primary cultured rabbit synoviocytes were long spindle fiber-like and coincided with the fibroblast-like synoviocytes. Moreover, cells were positive for Vimentin. In conclusion, fibroblast-like synoviocytes from rabbits with adjuvant arthritis are successfully isolated and cultured by the tissue explant method. [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) cell culture model is a system that co-culture carriers with 3D structural materials and different types of cells in vitro to simulate the microenvironment in vivo . This novel cell culture model has been proved to be close to the natural system in vivo . In the process of cell attachment, migration, mitosis and apoptosis, it could produce biological reactions different from that of monolayer cell culture. Therefore, it can be used as an ideal model to evaluate the dynamic pharmacological effects of active substances and the metastasis process of cancer cells. This paper compared and analyzed the different characteristics of cell growth and development under two-dimensional (2D) and 3D model culture and introduced the establishment method of 3D cell model. The application progress of 3D cell culture technology in tumor model and intestinal absorption model was summarized. Finally, the application prospect of 3D cell model in the evaluation and screening of active substance was revealed. This review is expected to provide reference for the development and application of new 3D cell culture models.

Conventional tumor culture models include two-dimensional tumor cell cultures and xenograft models. The former has disadvantages including lack of tumor heterogeneity and poor clinical relevance, while the latter are limited by the slow growth, low engraftment successful rate, and high cost. In recent years, in vitro three-dimensional (3D) tumor models have emerged as the tool to better recapitulate the spatial structure and the in vivo environment of tumors. In addition, they preserve the pathological and genetic features of tumor cells and reflect the complex intracellular and extracellular interactions of tumors, which have become a powerful tool for investigating the tumor mechanism, drug screening, and personalized cancer treatment. 3D tumor model technologies such as spheroids, organoids, and microfluidic devices are maturing. Application of new technologies such as co-culture, 3D bioprinting, and air-liquid interface has further improved the clinical relevance of the models. Some models recapitulate the tumor microenvironment, and some can even reconstitute endogenous immune components and microvasculature. In recent years, some scholars have combined xenograft models with organoid technology to develop matched in vivo / in vitro model biobanks, giving full play to the advantages of the two technologies, and providing an ideal research platform for individualized precision therapy for specific molecular targets in certain subtypes of tumors. So far, the above technologies have been widely applied in the field of colorectal cancer research. Our research team is currently studying upon the application of patient-derived tumor cell-like clusters, a self-assembly 3D tumor model, in guiding the selection of postoperative chemotherapy regimens for colorectal cancer. A high modeling success rate and satisfactory results in the drug screening experiments have been achieved. There is no doubt that with the advancement of related technologies, 3D tumor models will play an increasingly important role in the research and clinical practice of colorectal cancer.

Liver fibrosis incidence and adverse outcomes are high; however, there are no known chemical drugs or biological agents that are specific and effective for treatment. The paucity of a robust and realistic in vitro model for liver fibrosis is one of the major causes hindering anti-liver fibrosis drug development. This article summarizes the latest progress in the development of in vitro cell models for liver fibrosis, with a focus based on the analysis of induction and activation of hepatic stellate cells, cell co-culture, and 3D model co-construction, as well as concurrent potential methods based on hepatic sinusoidal endothelial cell establishment.

Objective: To explore the effect of alpha -linolenic acid (ALA) on human oocytes maturation and the mitochondrial DNA (mtDNA) copy numbers of oocyte. Methods: A total of 441 morphologically normal immature oocytes were recruited from 273 infertile women underwent ICSI in our Center from October 2014 to October 2015. There were 179 oocytes at M I stage and 262 oocytes at G V stage. Oocytes with the same proportion of M I and G V were randomly divided into 5 groups, and in vitro cultured for 24 hours in the medium with ALA at different level [0 (as control), 10 jxmol/L, 50 jxmol/L, 100 jxmol/L, 200 jxmol/L]. The oocyte maturation was observed, and the mtDNA copy numbers in the ALA 0 jxmol/L (as control) group and the ALA 50 jxmol/L group were determined by real - time PCR. Results: ① The total maturation rate of the ALA 50 jxmol/L group (70.1% ) was significantly higher than that of the control group (42.1% ) and the ALA 200 jxmol/L group (34.1% , both P 〈0.05).② The total maturation rate of G V in the ALA 50 jxmol/L group (64.4% ) was significantly higher than that in the control group (28.9% ) and the ALA 200 jxmol/L group (18.2% , both P 〈 0 .0 5 ) .③ The mtDNA copy number of in vitro matured oocytes in the ALA 50 jxmol/L group (3.64x107±1.85x106) was significantly higher than that in the ALA 0 jxmol/L group (3 .4 2 x106±1.95x105, P 〈0.05). Conclusions: 50 jxmol/L ALA can increase the mtDNA copy number of the in vitro matured human oocyte, suggesting that ALA can promote the in vitro maturation of human oocyte. [ABSTRACT FROM AUTHOR]

Objective To optimize and establish the methodology for culturing and inducing differentiation of mouse preadipocytes 3T3-L1. Methods The mouse cells 3T3-L1 were incubated in DMEM medium contained with 10% FBS, during which the incubation medium was refreshed every 2 to 3 days. Two methods were used to introduce differentiation, including three- drug combination group and four- drug combination group. The protocol of medium Ⅰ in three- drug combination group including insulin 10 mg/L, IBMX 0.5 mmol/L and DEX 1.0 μmol/L. The protocol of mediumⅠin fourdrug combination group including indometacin 0.1 mmol/L based on those of three-drug combination group. Both of them were incubated for 2 days and continuous for 2 times. And medium Ⅱ included insulin 10 mg/L for 2-day culturing and continuous for 2 times. Oil red O staining was used to observe the morphological changes of two groups of cells before and after treatment under inverted microscope. Results Mouse preadipocytes 3T3-L1 appeared in good conditions and grew in a paving stone fashion. These cells covered homogeneously the bottom of incubators, the culture medium refreshed every 2 days. The results of four-drug combination group were better than those of three-drug combination group. After three-drug combination induced differentiation, there was no significant change in cell morphology. Comparing with three- drug combination induced differentiation, four-drug combination was successfully achieved in over 90% of the cell inducing, which were round-shaped, with jacinth ester droplets by oil-red O staining. Conclusion We have optimized the method for culturing and inducing differentiation of mouse preadipocytes 3T3-L1 by adding indometacin on the basis of the three-drug combination induced differentiation. [ABSTRACT FROM AUTHOR]

Objective To establishing an immortal cell line of familial papillary thyroid carcinoma (FPTC), and explore a new approach for studying familial non-medullary thyroid carcinoma (FNMTC). Methods The specimen from a patient with FPTC was selected, separated, and the primary cells were cultured using DMEM/F12 medium (with TSH, T3, EGF and hydrocortisone). To inducing cell immortalization, the exogenous genes SV40T/TERT were transfected into cells by two ways. RT-PCR was used to detect the expressions of thyroid peroxidase (TPO), thyroid globulin (TG), thyroid stimulating hormone receptor (TSHR) and sodium/iodide co- transporter (NIS). Immunofluorescence method was used to detect the expressions of TPO and GPC3. In order to detect the genomic mutations, the peripheral blood DNA of the patient was extracted. The cell genome was detected. Results The FPTC cells adhered to the plate and showed an irregular polygon shape. The cells can stably grow for six months, FPTC-S (with SV40T transfected) passaged to p26, FPTC cells passaged to p23 and FPTC-ST (with SV40T/TERT transfected) passaged to p19. Both FPTC-S and FPTC-ST can stably express TPO, TG and TSHR in mRNA level. MLH1 R217C mutation existed in the peripheral blood of the patient, and BRAF V600E mutation existed in the primary cultured cells. Either the primary or the immortal cells showed MLH1 R217C mutation. Conclusion This study preliminarily established an immortal cell line of familial papillary thyroid carcinoma with MLH1 R217C and BRAF V600E mutations. This cell line provides a research model for studying these mutations in FPTC. [ABSTRACT FROM AUTHOR]

Objective Using abandoned white cells separated from preparation of blood products to cultivate NK cells in vitro, and to optimize the method of cultivation of allogeneic NK cells for clinical application. Methods Abandoned white cells separated from blood production were collected from 15 healthy donors. PBMCs were isolated from the abandoned white cells and cultured for 17 days using culture bottles as previously coated antibodies (group CD3 mAb was coated with CD3 mAb, group CD 16mAb was coated with CD16mAb, and group CD3 mAb+ CD16 mAb was coated with CD3 mAb and CD16 mAb). Flow cytometry was used to determine the ratio of CD3-CD56+ cells, expression of activated cell surface receptors, and secretion of IFN-γ. The anti-tumor cytotoxicity against K562 and Raji cells was determined using LDH cytotoxicity assay and flow cytometry. Results After expansion for 17 days, the proportions of CD3-CD56+ cells was (15.19±12.22)% in the group CD3 mAb, (83.63±10.63)% in the group CD16 mAb, (49.40±12.64)% in the group CD3 mAb+ CD16 mAb, and it was (16.34±10.51)% before expansion. The total number of NK cells was more than 109. The expression ratios of NK cell surface activated receptors NKp30 and NKp46 were significantly increased, while that of the NKG2D was not significantly changed. The NK cells after expansion showed high cytotoxicity activity against K562 cells, reaching up to(76.97±3.16)% when effector-cell-to-target-cell ratio (E∶T ratio) was 40∶1. Conclusions NK cells can be obtained from abandoned white cells after cultivation for 17 days, with a purity up to 90% and total cell number of more than 109. Their activity was reinforced, the anti-tumor cytotoxicity activity was increased, and may meet the standard of clinical therapeutic application. [ABSTRACT FROM AUTHOR]

Objective: To develop an efficient method for isolation and culture of mouse salivary gland stem cells. Methods: Salivary gland stem cells were isolated and cultured from 7~8 week old C57BL/6J mice salivary gland. Inverted microscope was used to observe the cells' morphology; Growth curve was determined by cell counting; the expression of laminin was analyzed by Immunofluorescence. Results: After two days in culture, small aggregates of cells (salispheres) will be apparent in the cultures. Salispheres will continue to grow in size over a period of four days in culture. The diameter is about 100 μm and there are no obvious spikes around the salispheres. After seven days in culture, obvious spikes occurred. The morphology of the salispheres after ten days was different from those in four days; the salispheres have a trend to differentiation. Growth curve show the number of the salispheres was increased in a time-dependent manner. At the same time, the diameter of the salispheres also increased significantly. These results demonstrated that the salivary gland stem cells had high proliferation ability. Immunofluorescence results showed that salivary stem cells began to express LN after 2 days in culture, and the level of LN was increased significantly after 4 days in culture. Conclusion: The results of this study demonstrated that the mouse salivary gland stem cells from the mice salivary gland through this improved method possessed the biological characteristics of adult stem cells. These cells had the ability to undergo both self-renewal and multi-directional differentiation. [ABSTRACT FROM AUTHOR]

Objective To establish a method for in vitro culture of plasmacytoid dendritic cell (pDC). Methods Umbilical cord blood (40 ml) was collected from healthy parturients in the First Affiliated Hospital of Hunan University of Chinese Medicine, and cord blood mononuclear cell (CBMC) were isolated. The CBMC were cultured for 7 days with RPMI 1640 complete medium containing rh Flt3-ligand (Flt3-L) (100 ng/ml) and rh interleukin (IL)-3 (10 ng/ml), and the medium was half changed every 2 days. On the eighth day, CpG ODN (2 μg/ml) was added to the cells, and the attached cells and supernatant were collected 24 h later for flow cytometry and interferon (IFN)α measurement, respectively. On days 1, 3, 5, 7, and 8 of cell culture, the morphological changes of pDC were observed. Results After 2 h of culture, the CBMC showed circular, flat morphology. Twenty-four hours later, the cells began to adhere to the wall, with extended cytoplasm and increased volumes, and they became round and translucent, with scattered small colonies. On days 3-4 of culture, the cell volume continued increasing; most cells were round, and some had small protrusions; few cells were spindle-, tadpole-, star- or irregularly shaped; the number and volumes of colonies increased substantially. On days 5-8 of culture, the number of colonies and the number of cells in colonies gradually decreased, and suspended cells that were round or had small protrusions gradually increased in the medium. The cells expressing CD123, BDCA-2, and BDCA-4, which were considered pDC, were detected by flow cytometry. Flow cytometry revealed that the proportion of pDC in CBMC increased during the culture: increasing from 1.08% at the beginning of culture to 5.32% on day 4, and finally reaching a peak of 19.8% on day 8. On day 8, the level of IFNα in pDC culture supernatant was(11 302.61±1745.31) pg/ml. Conclusion pDC can be successfully induced in vitro by rh Flt3-L combined with IL-3 from human umbilical CBMC. [ABSTRACT FROM AUTHOR]

HBV is a major health problem faced by human beings. The study of the mechanism of HBV infection has been a key point in this field. To carry out the related research on HBV, establishment of simple and available in vivo and in vitro infection models is the trend. Due to the close relationship between tree shrews and human/primates, the tree shrew model of HBV infection has attracted more and more attention. This review summarizes recent research, both at home and abroad, about in vivo and in vitro HBV infection in tree shrews, especially the research progress in in vitro culture of primary hepatocytes of tree shrews, and looks to the future research directions as well. [ABSTRACT FROM AUTHOR]

Objective: To investigate an appropriate method for isolating and assessing EPCs from mice bone marrow. This could offer a better support to further research on EPCs. Methods: BM mononuclear cells were isolated from mice bone marrow by density gradient centrifugation and were cultured in conditioned medium. Early endothelial progenitor cells and late endothelial progenitor cells were cultured by adherent culture method. Early EPCs were analyzed by FACS on days 0, 6 and 10. EOCs were analyzed after 8 weeks. Results: EEPCs and EOCs were cultured via the adherent culture of bone marrow, the percentage with EEPC phenotypes (CD34+/CD133+/VEGFR2+), increased from 0.08 %to 70 %when cultured for 7 days in the EPC medium. EOC appeared in about 3 to 4 weeks, in 5 to 8 weeks showed exponential growth, which expressed CD31 and VEGFR2. Conclusion: The primary establishment of isolation, cultivation and identification of EPCs from mice bone marrow, indicates that EPCs can be isolated and identified in vitro. It will be the basis for further research and clinical applications. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) infection has become the main cause of hepatocellular carcinoma. A series of cell models for HBV infection are established to lay a good foundation for the research on the pathogenesis and treatment of HBV. In this review, we summarize culture models for a single type of cells, co-culture models for multiple types of cells, and other animal models, which are mainly for HBV transfection and infection. We also discuss the methods of cell model construction for HBV infection and their virological characteristics. It provides reliable evidence of models for scientific interpretation of advantages of traditional Chinese medicine in the prevention and treatment of chronic hepatitis B. [ABSTRACT FROM AUTHOR]

Objective To optimize primary cultures techniques of isolating neonatal rat cardiomyocytes and to compare three different methods of extracting β3-adrenergic receptor（β3-AR）membrane protein from cultured neonatal rat cardiomyocytes. Methods TypeⅡ collagen and differential velocity adhesion were used to collect primary cardiomyocytes. Total protein method, ultracentrifugation method, extract kit method were used to isolate cardiomyocytes β3-AR membrane proteins. The BCA method was applied for protein quantification. Relative content of β3-AR membrane protein and GADPH in the sample were examined by western blot. Results Optimizing culture and isolation skills can produce a great quantity of cardiomyocytes in high concentration.The kit method acquired a higher level of protein concentration（8.26±0.29）g/L than total protein method（5.12±0.47）g/L does than ultracentrifugation method（3.20±0.37）g/L does all of which were with significant difference（P < 0.05）. The concentration of β3-AR membrane protein was higher if obtained by kit method（0.22± 0.05）than ultracentrifugation method（0.09±0.03）than total protein method (0.01±0.01) with significant difference（P < 0.05）. Conclusion optimizing methodology can obtain abundant myocardial cells in high concentraion. The kit method of isolating primary cultured β3-AR membrane proteins result in improved concentration and specificity of membrane protein. [ABSTRACT FROM AUTHOR]

Objective To establish a three - dimensional (3D) cell culture model for gallbladder carcinoma (GC) cells and to investigate the kiiling effect of a novel nano - drug delivery system on GC cells and its action mechanism. Methods Two - dimensional (2D) and 3D cell culture models for GC GBC - SD cells were established, and the normal cell toxicity of nano - drug delivery system ( CP24, folate recep-tor - mediated targeting poly ( epsilon - caprolactone ) ) and the inhabitation rate of GC cells exposed to 5 - fluorouracil ( 5 - FU ) carried by CP24 were determined by MTT assay, HE staining, flow cytometry (FCM), and HoechstPI staining. Results CP24 showed no significant cytotoxicity in liver and kidney cells, according to the result of HE staining. CP24 showed no significant kiiling effect on GC cells, according to the result of FCM. Given a 5 - FU concentration of 100 |ig/ml, the kiiling and inhibitory effects of both 5 - FU - CP24 and 5 - FU on GBC - SD cells were lower when using the 3D cell culture model than when using the 2D cell culture model; whether using a 2D or 3D cell culture model, 5 - FU - CP24 had a significantly stronger kiiling effect on GBC - SD cells than 5 - FU ( P < 0. 05 ) . 5- FU - CP24 mainly led to apoptosis in GBC - SD cells, while 5 - FU mainly led to necrosis, as shown by HoechstPI staining. Conclusion 3D cell culture can reflect the intracellular microenvironment in vivo more accurately. Intercellular matrix may affect the cellular sensitivity to drugs. 5 - FU can kiil more GC cells when carried by a nano - drug delivery system. This degradable nano - drug delivery system has good drug delivery per-formance, sustained drug release, and high biosafety and holds promise for application in chemotherapy for GC. [ABSTRACT FROM AUTHOR]

Based on the current study of the influence of mechanical factors on cell behavior which relies heavily on experiments in vivo , a culture chamber with a large uniform strain area containing a linear motor-powered, up-to-20-Hz cell stretch loading device was developed to exert mechanical effects on cells. In this paper, using the strain uniformity as the target and the substrate thickness as the variable, the substrate bottom of the conventional incubation chamber is optimized by using finite element technique, and finally a new three-dimensional model of the incubation chamber with "M" type structure in the section is constructed, and the distribution of strain and displacement fields are detected by 3D-DIC to verify the numerical simulation results. The experimental results showed that the new cell culture chamber increased the accuracy and homogeneous area of strain loading by 49.13% to 52.45% compared with that before optimization. In addition, the morphological changes of tongue squamous carcinoma cells under the same strain and different loading times were initially studied using this novel culture chamber. In conclusion, the novel cell culture chamber constructed in this paper combines the advantages of previous techniques to deliver uniform and accurate strains for a wide range of cell mechanobiology studies.

Objective: To explore effect of lentivirus-mediated complement C3 silencing vector on the osteogenic ability of human B lymphocyte Raji-osteoblast cell line MG63 co-culture system and its mechanism., Methods: The lentiviral complement C3 silencing vector was constructed and transfected into human B lymphocyte Raji to establish in vitro Raji-osteoblast cell line MG63 co-culture system. The cells were divided into blank control group (without special treatment), complement C3 silencing group (lentiviral complement C3 silencing vector transfection co-culture system) and model group(lentiviral vector transfection co-culture system). The expression of complement C3 in each group was detected by reverse transcription-polymerase chain reaction(RT-PCR) and Western-Blot at 24 h after culture, proliferation of MG63 cells was detected by CCK-8 at 0, 3, 6, 12, 24, 48 and 72 h, apoptosis of MG63 cells in each group was detected by flow cytometry at 24 h after culture, and alkaline phosphatase(AKP) activity of MG63 cells in each group was detected by AKP detection kit, and osteoprotegerin (OPG ) protein expression of MG63 cells in each group was detected by Western-Blot method., Results: RT-PCR results showed that the expression level of C3 in complement C3 silencing group was lower than that in blank control group and model group, Western-Blot results showed that expression of C3 in complement C3 silencing group was lower than that in blank control group and model group, CCK-8 results showed that there was no difference in proliferation ability of MG63 among complement C3 silencing group and blank control group and model group at 3 and 6 h after culture;at 12 h after culture, proliferation ability of MG63 cells with C3 silencing was higher than that of blank control group and model group;at 24, 48 and 72 h after culture, proliferation ability of MG63 cell line with complement C3 silencing group were higher than that of blank control group and model group. Flow cytometry resluts showed that apoptosis of proliferation ability of MG63 cell line with complement C3 silencing group was lower than that of blank control group and model group;AKP detection kit suggested that AKP activity in complement C3 silencing group was higher than that in blank control group and model group, Western-Blot results showed that expression level of OPG protein in complement C3 silencing groupwas higher than that in blank control groupand model group., Conclusion: Silencing of complement C3 could enhance bone formation ability of osteoblast MG63, and it takes time to accumulate this ability. Complement C3 may affect osteogenesis by altering OPG / RANKL / RANK axis.

Salivary glands are important organs in the oral and maxillofacial region. Environment and genetic factors may cause salivary gland tumors or non-neoplastic diseases, but the mechanisms of those diseases are still unclear. One of the important reasons is the short of researching media and model. As a new technique and research model, organoids have been widely used in the research of various diseases. Organoid culture plays a bridging role between two-dimensional cell culture and living animal models, and it is also the most promising translational research model that could connect the clinical research to basic research. This review will discuss the recent development of organoid techniques in the culture of normal salivary glands and salivary gland tumors, also their applications and challenges in tissue engineering, etiological research, and tumor therapy.

Objective To provide an important scientific basis for verifying the in vitro anti-HBV activity of single herb Rabdosia Serra. Methods 50%ethanol extract of Rabdosia Serra was dispensed as certain concentrations. The HepG2.2.15 cel-lular model was adopted to conduct the cellular toxicity test and HBsAg and HBeAg enzyme-linked immunosorbent ssay(ELISA). Results IC50 was 4.275 mg/mL,IC50 of crude extract of Rabdosia Serra on HBsAg was 2.250 mg/mL, which on HBeAg was 2.150 mg/mL, the therapeutic index(TI)<2. Conclusion The crude extract of Rabdosia Serra has the effect for in vitro inhibiting HBV. [ABSTRACT FROM AUTHOR]

Many studies have shown that Chinese herb extracts could treat patients who suffer hepatitis C. However, the clinical efficacy is limited due to the use of traditional Chinese medicine (TCM) compounds. Because each drug contains multiple active substances, some of which may even act against each other in vivo, it is hard to clarify the mechanism of treatment. The current research on pseudotyped hepatitis C virus (HCV), replicon, and cell culture system is summarized, and the advances in antiviral research on the monomers or active components of Chinese herbs in vitro based on HCV RNA models are reviewed, thus providing new ideas for TCM treatment of hepatitis C. [ABSTRACT FROM AUTHOR]

In the process of xenobiotic toxicity prediction and risk assessment, in vitro cell culture models possess high practical application value. With the rapid development of biological technologies such as three-dimensional (3D) bio-printing, organoid culture and organ-on-a-chip systems, in vitro cell culture models have made great progress. Sharing the similarities in structure, function and the physiological environment with tissues or organs in vivo , hazard identification and dose-response analysis based on 3D cell culture models provide access to more accurate toxicity data as a theoretical basis for risk assessment and risk management of chemicals. This review summarizes the establishment of three typical 3D cell culture models, i.e., human cell line-based co-culture model, 3D-printed scaffold-based cell culture model and organoids, and their application in toxicity tests of xenobiotics.

Objective: To investigate the effect of vitamin D3 to platelet activation by tumor cell culture medium., Methods: The peripheral blood platelets of BALB/c mice were isolated. The platelets were activated in 4T1 culture fluid for 24 h. The platelets were divided into 7 groups: control group, activation group, 1 nmol/L vitamin D3 group, 10 nmol/L vitamin D3 group, 50 nmol/L vitamin D3 group, 100 nmol/L vitamin D3 group, and positive drug (0.1 μmol/L eptifibatide) group. CCK-8 assay was used to detect the platelet proliferation at 24, 48 and 72 h. Flow cytometry was used to detect the expression of CD61 and CD62p and receptor for advanced glycation end products (RAGE) at 24, 48 and 72 h. ELISA was used to detect the level of platelet-endothelial cell adhesion molecule-1 (PECAM-1) at 24, 48 and 72 h., Results: The CD41 + /CD61 + and CD41 + /CD62p + ratio, PECAM-1 content and RAGE expression of platelets in activated group were all significantly increased as compared with those in control group (P<0.05). Compared with the activated group, the platelet proliferation, proportion of CD41 + /CD61 + and CD41 + /CD62p + , content of PECAM-1 and RAGE expression in vitamin D3 groups were all decreased (P<0.05)., Conclusion: Vitamin D3 shows antiplatelet effect and can inhibit platelet proliferation and activation.

Breast cancer is the most common cancer in women. At present, the in vivo model and traditional cell culture are mainly used in breast cancer researches. However, as high as 90% clinical trials are failed for drugs explored by the above two methods, due to the inherent species differences between humans and animals, as well as the differences in the tissue structure between organs and cells. Therefore, organoid three-dimensional culture is emerging. As a new tumor research model, organoid, a three-dimensional cell complex with spatial structure, has broad application prospects, such as precision medicine, organ transplantation, establishment of refractory disease model, gene therapy and drug research and development. Therefore, organoid is considered as one of the ideal carriers for life science research in the future. Breast cancer, a heterogeneous disease with complex phenotypes, has a low survival rate. Breast cancer organoid can reproduce many key features of human breast cancer, thus, the construction of organoid biological library of breast cancer will provide a new platform for studying the occurrence, development, metastasis and drug resistance mechanism of breast cancer. In this review, we systematically introduce the culture conditions of organoids and their application in breast cancer related research, and the application prospect of organoids.

With the advantages of low immunogenicity and long half-life, human monoclonal antibody has become an indispensable biological agent in vivo. Immortalization of human B cells is a potential and effective method to obtain natural human antibody library, which can provide a rich source for the preparation of human monoclonal antibodies. As there are urgent problems to be solved in each platform, the preparation of antibodies based on human B cell immortalization is still limited to the laboratory research stage. At present, there is a lack of a systematic review to clarify the advantages and disadvantages of the existing human B cell immortalization antibody preparation platform and its feasibility analysis. This paper reviews the research on the preparation of human monoclonal antibody based on human B cells immortalization, and describes an in vitro cell culture method, in which hCD40L vesicles are used instead of feeder cells, in order to provide references for the further development of human monoclonal antibody preparation technology.

Objective: To develop a new method to activate and expand human NK cells ex vivo by using sodium hyaluronate as a major activating agent and to explore its related mechanism., Methods: Mononuclear cells were isolated from 3 samples of peripheral blood from three healthy donors. New NK cell culture method and the control method were used to culture NK cells from each samples separately for 14 days. Flow cytometry was used to analyze the ratio of NK cells and CD69 expression. To measure the in vitro cytotoxicity of NK cells cultured by the two methods, the K562 cells were used as the targeting cells and flow cytometry combined with CFSE marker was used as the testing method., Results: After culturing for 14 days, the number of NK cells obtained by new culture method for NK cells expanded by 188.63±3.83 times while the number of NK cells cultured by control method expanded by 152.77±5.77 times. The ratio of NK cells in new cell culture method was above 90%, while the ratio of NK cells in control method was about 70%. The ratio of CD69+ NK cells in new cell culture method was 32.37%±3.22%, while the ratio of CD69+ NK cells in control method was 17.29%±3.79%. The results of cytotoxicity experiment in vitro showed that NK cells cultured by the new method had a higher killing ability to the target cells as compared with NK cells cultured by the control method., Conclusion: New NK cell culture method using sodium hyaluronate as a major activating agent can expand NK cells more efficiently as compared with the cells cultured by control method, which may be related to the direct and/or indirect activation of sodium hyaluronate to NK cells, further causing the dominant expansion of the NK cells.

Objective: To compare the curative effect of mesenchymal stem cells derived from human Wharton's Jelly(WJ-MSC) or adipose(AD-MSC) culture supernatant on endothelial cells angiogenesis. Methods: WJ-MSC and AD-MSC were isolated, identified, and the culture supernatant of stem cells was collected.The WJ-MSC or AD-MSC supernatant co-cultured with the endothelial cells. The expression levels of pro-angiogenic and anti-angiogenic genes of endothelial cells were assessed using qRT-PCR analysis, and the effects of stem cell culture supernatant on angiogenesis were evaluated by performing a tube formation assay in vitro. Results: After adding WJ-MSC and AD-MSC culture supernatant, the expression levels of pro-angiogenic genes in endothelial cells were upregulated, and the expression levels of anti-angiogenic genes were downregulated significantly in both experimental groups compared to the control group ( P< 0.01), and tube formation of endothelial cells was also significantly increased in both experimental groups as determined by the increase of the tube length ((43.2±9.2) mm vs (94.3±13.2)mm, (86.1±7.2)mm, P< 0.01). Conclusion: The results showed that AD-MSC culture supernatant can promote endothelial cells angiogenesis and its curative effect is similar to that of WJ-MSC.

Objective: To explore a new strategy for constructing three-dimensional dermoid tissue in vitro by using cell sheets technology., Methods: Rabbit bone marrow mesenchymal stem cells (rBMSCs) were isolated from bone marrow of New Zealand white rabbits and cultured by whole bone marrow adherent method. Human dermal fibroblasts (HDFs) were cultured and passaged in vitro . The 2nd generation rBMSCs and the 3rd generation HDFs were cultured in a culture dish for 2 weeks with cell sheets conditioned medium respectively to obtain a monolayer cell sheets. Human umbilical vein endothelial cells (HUVECs) were inoculated on rBMSCs sheet to construct pre-vascularized cell sheet. During the culture period, the morphological changes of the cell sheet were observed under an inverted phase contrast microscope. At 1, 3, 7, and 14 days, HE staining and CD31 immunofluorescence staining were performed to observe the cell distribution and microvascular network formation. The rBMSCs sheet was used as control. The pre-vascularized cell sheet (experimental group) and rBMSCs sheet (control group) cultured for 7 days were placed in the middle of two HDFs sheets, respectively, to prepare three-dimensional dermoid tissues. After 24 hours of culture, CD31 immunofluorescence staining and collagen type Ⅰ and collagen type Ⅲ immunohistochemical stainings were performed to evaluate cell distribution and collagen expression., Results: HDFs and rBMSCs sheets were successfully prepared after 2 weeks of cell culture. After inoculation of HUVECs on rBMSCs sheet for 3 days, HUVECs could be seen to rearrange on rBMSCs sheet and forming vacuoles. The reticular structure was visible at 7 days and more obvious at 14 days. The formation of vacuoles between the cell sheets was observed by HE staining, and the vacuoles became more and more obvious, the thickness of the membranes increased significantly with time. CD31 immunofluorescence staining showed the microvascular lumen formation. However, only the thickness of rBMSCs sheet increasing was observed, with no changes in cell morphology or cavitation structure. The three-dimensional dermoid tissue observation showed that the endothelial cells in the experimental group were positive expressions, and the rBMSCs, HDFs, and HUVECs cells were arranged neatly. The endothelial cells were negative expressions and randomly arranged in the control group. The collagen type Ⅰ and collagen type Ⅲ were positive expression in the experimental group and the control group. But compared with control group, experimental group presented a "honeycomb" network connection, where the matrix was distributed regularly, and cells were arranged tightly. The difference in the expression of collagen type Ⅰ and collagen type Ⅲ between the experimental group and the control group was not significant ( P >0.05)., Conclusion: Three-dimensional dermoid tissue is successfully constructed by using cell sheet technology. The cell matrix distribution of the pre-vascularized cell sheet constructed by HUVECs and rBMSCs sheet is relatively regular, which has the potential to form tissue engineered dermis.