PURPOSE: The incidence of acute lung injury (ALI) in severe trauma patients is 48% and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%. Alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s) are the key cells in the repair of injured lungs as well as fetal lung development. Therefore, the purification and culture of AEC1s and AEC2s play an important role in the research of repair and regeneration of lung tissue. METHODS: Sprague-Dawley rats (3-4 weeks, 120-150 g) were purchased for experiment. Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells, and then magnetic bead cell sorting was performed to isolate T1α positive cells as AEC1s from the single-cell suspension by using polyclonal rabbit anti-T1a (a specific AEC1s membrane protein) antibodies combined with anti-rabbit IgG microbeads. Afterwards, alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining T1α-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads. Cell purity was identified by immunofluorescence staining and flow cytometry. RESULTS: The purity of AEC1s and AEC2s was 88.3% ± 3.8% and 92.6% ± 2.7%, respectively. The cell growth was observed as follows: AEC1s stretched within the 12-16 h, but the cells proliferated slowly; while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days. CONCLUSION AEC1s and AEC2s sorted by this method have high purity and good viability. Therefore, our method provides a new approach for the isolation and culture of AEC1s and AEC2s as well as a new strategy for the research of lung repair and regeneration.
Alveolar Epithelial Cells/cytology* ; Animals ; Cell Culture Techniques ; Cell Separation/methods* ; Immunoglobulin G/metabolism* ; Lung ; Magnetic Phenomena ; Rats ; Rats, Sprague-Dawley

In this study, we improved the culture method of mouse hippocampal primary microglia to obtain hippocampal ramified microglia with high activity and purity, which were resemble to the resting status of normal microglia in healthy brain in vivo. Hippocampal tissue was excised from 2-4-week-old SPF C57BL/6J mice and cut into pieces after PBS perfusion, and then manually dissociated into the single-cell suspension by using Miltenyi Biotec's Adult Brain Dissociation Kit. The tissue fragments such as myelin in the supernatant were removed by debris removal solution in the kit. The cell suspension was incubated with CD11b immunomagnetic beads for 15 min at 4 °C. To obtain high-purity microglia, we used two consecutive cell-sorting steps by magnetic activated cell sorting (MACS). After centrifugation, the cells were resuspended and seeded in a 24-well culture plate. The primary microglia were cultured with complete medium (CM) or TIC medium (a serum-free medium with TGF-β, IL-34 and cholesterol as the main nutritional components) for 4 days, and then were used for further experiments. The results showed that: (1) The cell viability was (56.03 ± 2.10)% by manual dissociation of hippocampus; (2) Compared with immunopanning, two-step MACS sorting allowed for efficient enrichment of microglia with higher purity of (86.20 ± 0.68)%; (3) After being incubated in TIC medium for 4 d, microglia exhibited branching, quiescent morphology; (4) The results from qRT-PCR assay showed that the levels of TNF-α, IL-1β and CCL2 mRNA in TIC cultured-microglia were similar to freshly isolated microglia, while those were much higher in CM cultured-microglia after incubation for 4 d and 7 d (P < 0.05). Taken together, compared to the conventional approaches, this modified protocol of mouse hippocampal primary microglia culture by using MACS and TIC medium enables the increased yield and purity of microglia in the quiescent state, which is similar to normal ramified microglia in healthy brain in vivo.
Animals ; Cell Culture Techniques ; methods ; Cell Separation ; methods ; Cells, Cultured ; Hippocampus ; Magnetics ; Mice ; Mice, Inbred C57BL ; Microglia ; cytology

Human lipocalin 6 (hLCN6) is an epididymis-specific secretory protein. It binds to sperm and plays important role in sperm maturation. To explore the feasibility for isolating spermatozoa from mixed cells using anti-hLCN6 monoclonal antibody-conjugated immunomagnetic beads (anti-hLCN6 IMBs) and establish a new method for the separation of sperms from mixed stains, 2 sets of 30 cases of cell mixture suspensions and stains containing different proportions of sperm and epithelial cells were prepared. Biotin-labeled anti-hLCN6 monoclonal antibody (mAb) was incubated with the cell mixtures, and the spermatozoa were then isolated with avidin-coated IMBs. Sperm DNA was extracted and analyzed by PCR-STR typing. Differential lysis was also conducted to compare the effect of the two different isolation methods. The dissociation constant (Kd) of anti-hLCN6 mAb was 3.47×10⁻⁹ mol/L measured by ELISA. Western blotting and immunofluorescence assays showed that hLCN6 was detectable on sperm cells and mainly located on the post-acrosomal region of the sperm head, but not in epithelial cells. Anti-hLCN6 IMBs could capture and separate the sperm cells successfully. Microscopic observation showed that the IMBs could bind to the head of sperm specifically. The success rate of STR typing (more than 13 STR loci, RFU>200) was 90% when the number of sperm cells was 10³/mL and 100% when the sperm cells number was equal to or more than 10⁴/mL. When the number of sperm cells was 10³/mL, 10⁴/mL and 10⁵/mL in mixed stain samples, the success rate of STR typing were 40%, 90% and 100%, respectively. Taken together, the anti-hLCN6 immunomagnetic beads (IMB) method described here could be effective for the isolation of sperm from mixed cells, and the success rate was higher than that of the traditional differential lysis strategy. IMB sorting is a simple and efficient method for the separation of sperms from sperm and epithelial cell mixture, and can be utilized as a supplementary method for forensic mixture samples analysis in sexual assault cases.
Cell Separation ; DNA ; Humans ; Immunomagnetic Separation ; Lipocalins ; Male ; Polymerase Chain Reaction ; Spermatozoa

Basic research in life science and medicine has dug into single cell level in recent years. Single-cell analysis offers to understand life from diverse perspectives and is used to profile cell heterogeneity to investigate mechanism of diseases. Single cell technologies have also found applications in forensic medicine and clinical reproductive medicine, while the techniques are rapidly evolving and have become more and more sophisticated. In this article, we reviewed various single cell isolation techniques and their pros and cons, including manual cell picking, laser capture microdissection and microfluidics, as well as analysis methods for DNA, RNA and protein in single cell. In addition, we summarized major up-to-date single cell research achievements and their potential applications.
Animals ; Cell Separation ; DNA ; Laser Capture Microdissection ; RNA ; Single-Cell Analysis

OBJECTIVE: To establish the STO cell lines expressing green fluorescent protein (GFP) and mouse leukemia inhibitory factor (LIF) , and try to culture the mouse embryonic stem cells (mESCs) by using the established STO-GFP-mLIF cells as the feeder layer. METHODS: The lentiviral particles containing GFP and mLIF and puromycin-resistance gene were constructed and transduced into STO cell lines. The cell lines stably expressing GFP and mLIF genes were screened out. The expression level of the inserted exogenous LIF gene was tested by Western blot and ELISA. The STO-GFP-mLIF cells were treated with different concentrations of mitomycin C (5, 10, 15, 20 µg/ml) for different time (1.5, 2.5, 3, 3.5 hours) to prepare feeder layers and the cell proliferation level on feeder layer was observed. Mouse embryonic stem cells were cultured on mitomycin C-treated feeder layer and the growth of cell colonies was observed. RESULTS: The expression level of LIF protein in STO-GFP-mLIF cells was up-regulated, as compared with STO cells (P＜0.05). It was confirmed that the optimal concentration and time for inhibiting the proliferetion of STO-GFP-mLIF cells by mitomycin C were 10 µg/ml and 3 hours respectively. The observation also found that the embryonic stem cells could develop into typic "birdnest" shaped stem cell colony on mitomycin C-treated feeder layer. CONCLUSION The stable STO cell lines effectively expressing green fluorescent protein and mouse leukemia inhibitory factor have been established successfully, which can maintain the undifferentiated state of mouse embryonic stem cells.
Animals ; Cell Differentiation ; Cell Line ; Cell Separation ; Embryonic Stem Cells ; Feeder Cells ; Green Fluorescent Proteins ; Leukemia Inhibitory Factor ; Mice

White matter damage, characterized by demyelination due to the damage of oligodendrocyte precursor cells (OPCs), is the most common type of brain damage in preterm infants. Survivors are often subject to long-term neurodevelopmental sequelae because of the lack of effective treatment. In recent years, it has been found that cell transplantation has the potential for the treatment of white matter damage. OPCs are frequently used cells in cell transplantation therapy. With abilities of migration and myelinization, OPCs are the best seed cells for the treatment of white matter damage. Several studies have found that OPCs may not only replace impaired cells to reconstruct the structure and function of white matter, but also inhibit neuronal apoptosis, promote the proliferation of endogenous neural stem cells, and enhance the repairment of the blood-brain barrier. However, the clinical application of OPC transplantation therapy faces many challenges, such as the effectiveness, risk of tumorigenesis and immune rejection. With reference to these studies, this article reviewed the development of myelination, the obtainment of OPCs, the therapeutic mechanism as well as application research, and analyzed the current challenges of OPC transplantation, in order to provide a new direction for clinical treatment of white matter damage in preterm infants.
Cell Separation ; Demyelinating Diseases ; therapy ; Humans ; Infant, Newborn ; Infant, Premature ; Oligodendrocyte Precursor Cells ; transplantation ; White Matter ; pathology

BACKGROUND AND OBJECTIVES: Lack of understanding of the interplay between hematopoietic stem cells (HSCs) and the immune system has severely hampered stem cell research. Programmed death-1 (PD-L1) has been reported on parenchymal cells in patients with chronically inflamed livers and found to play an essential role in T cell homeostasis regulation. However, the bidirectional interaction between HSCs and lymphocytes remains elusive. Here, we aimed to get more insight into circulating CD34+ HSCs PD-L1 expression and T cell apoptosis in chronic HCV infected patients. METHODS: CD34+ HSCs were isolated and purified by immunomagnetic separation. PD-L1 expression was analyzed by quantitative PCR and flow cytometry. Furthermore, co-culture experiments between CD34+ HSCs and T-lymphocytes were established. T-cell lymphocyte apoptosis in peripheral blood and in cultures was detected. RESULTS: CD34+ HSCs constitutively express low levels of PD-L1. Its expression is up-regulated in chronic HCV infected patients. Moreover, PD-L1 expression on circulating CD34+ HSCs enhanced T cell apoptosis in peripheral blood and co-culture. CONCLUSION: Our results suggest novel bidirectional interplay between HSCs and lymphocytes mediated by PD-L1 expression on CD34+ HSCs. PD-L1 expression correlated with T-cell lymphocyte apoptosis. This may contribute to immunomodulatory properties of HSCs which improves its use for allogeneic transplantation.
Apoptosis ; Coculture Techniques ; Flow Cytometry ; Hematopoietic Stem Cells ; Hepatitis C, Chronic ; Hepatitis, Chronic ; Homeostasis ; Humans ; Immune System ; Immunomagnetic Separation ; Liver ; Lymphocytes ; Polymerase Chain Reaction ; Stem Cell Research ; T-Lymphocytes ; Transplantation, Homologous

BACKGROUND: We carried out a questionnaire survey for laboratories performing human leukocyte antigen-crossmatch (HLA-XM) to provide a basis for laboratory standardization of HLA-XM tests in Korea. METHODS: The questionnaires were distributed to 51 HLA laboratories participating in the HLA-XM part of the HLA proficiency survey program organized by the Korean Society for Laboratory Medicine and replies from 50 laboratories were analyzed. The questionnaires included following items: 1) HLA-XM methods performed and annual number of tests, 2) types of the specimen and lymphocyte separation methods, 3) test procedures and reagents for complement-dependent cytotoxicity crossmatch (CDC-XM) and flow cytometry crossmatch (FCXM). RESULTS: The number of laboratories performing anti-human globulin (AHG) CDC-XM (47/49, 96%) and FCXM (30/50, 60%) was considerably increased compared to the 2005 survey (AHG CDC-XM, 35/43, 81%; FCXM, 7/44, 16%). As for the annual number of XM tests, more than 50% of the laboratories were low volume laboratories performing ≤50 tests, and only 10% of the laboratories were performing >500 tests. For cell isolation methods, negative selection was used by 43% (21/49) of laboratories performing CDC-XM. Number of cells reacted per 1 µL of serum varied among different laboratories in both CDC-XM (1,000–8,000) and FCXM tests (1,300-20,000). For the interpretation of FCXM, log fluorescence ratio (26/30, 87%) was more commonly used than channel shift values (5/30, 17%). CONCLUSIONS: Considerable variation is noted in both CDC-XM and FCXM methods performed by different laboratories. A continuous effort for laboratory standardization is needed to reduce inter-laboratory variation in the HLA-XM test results.
Cell Separation ; Flow Cytometry ; Fluorescence ; Humans ; Indicators and Reagents ; Korea* ; Leukocytes ; Lymphocytes

Objective: To search for the methods of isolating, purifying and culturing corpus cavernosal endothelial cells (CCECs) from SD rats, observe their growth characteristics, and providing seed cells for the study of erectile dysfunction (ED). METHODS: The corpus cavernosal tissue from the SD rat was digested with 0.1% elastase, followed by purification of CCECs with immunomagnetic beads. After further amplification, monoclonal CCECs were sorted out with the cloning cylinder and their morphological and proliferative characteristics were observed. The von Willebrand factor (VWF) in the CCECs was identified by immunofluorescence staining, the CD31 molecule detected by immumohistochemistry, the purity of the CCECs determined by flow cytometry, and the proliferation of the cells measured with CCK-8 and growth curves. RESULTS: After 7 days of purification and culture, the CCECs were fused into a monolayer under the inverted phase-contrast microscope, arranged like flagstones. The growth curves showed that the CCECs were in latency with a low growth rate at 1－2 days, in the logarithmic growth phase with a rapid rate at 3－4 days, and into the platform phase around the 6th day. VWF was positively expressed in the CCECs with much green fluorescence, and so was CD31 with a large number of brownish particles. The positive rate of the CCECs which were labelled with the VWF purified with magnetic beads combined with cloning cylinders was up to (91.9±3.75)%. CONCLUSIONS High-purity rat CCECs can be cultured successfully using immunomagnetic beads combined with cloning cylinders, with stable proliferation and passage in the endothelial cell medium.
Animals ; Cell Culture Techniques ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Endothelial Cells ; chemistry ; cytology ; physiology ; Erectile Dysfunction ; pathology ; Flow Cytometry ; Humans ; Immunomagnetic Separation ; Male ; Penis ; cytology ; Platelet Endothelial Cell Adhesion Molecule-1 ; analysis ; Rats ; Rats, Sprague-Dawley ; Sincalide ; analysis ; von Willebrand Factor ; analysis

Objective To develop an improved method for culturing Schwann cells(SCs) by using both enzyme digestion and explants-culture approaches and compared with traditional explants-culture method and general hemi-explants-culture method. Methods Bilaterally sciatic nerves and brachial plexus nerves were dissected from 3 to 5-day-old neonatal SD rats and explants-culture method,general hemi-explants-culture method,and improved enzyme digestion combined with explants-culture method were adopted to culture SCs,respectively. SCs were digested and passaged after 7 days in culture and counted under the microscope. The purity of SCs was identified by S-100 immunofluorescence staining. Results The SCs of improved method group grew fastest and the total number of cells obtained was(1.85±0.13)×10(6);the SCs of the hemi-explants-culture method group grew slower than the improved method group and the total number of cells obtained was (1.10±0.10)×10(6);the SCs of the explants-culture method group grew slowest and the total number of cells obtained was (0.77±0.03)×10(6).The total number of cells obtained showed significant difference among the three groups(P<0.01). Immunofluorescence staining showed that the SCs purity was (95.73±1.51)% in the improved method group,(84.66±2.68)% in the hemi-explants-culture method group,and (74.50±4.23)% in the explants-culture method group(P<0.01). Conclusion The improved enzyme digestion combined with explants-culture method can obtain sufficient amount of high-purity SCs in a short time and thus may be applied in further research on peripheral nerve regeneration.
Animals ; Animals, Newborn ; Brachial Plexus ; cytology ; Cell Culture Techniques ; Cell Separation ; methods ; Cells, Cultured ; Enzymes ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; cytology ; Sciatic Nerve ; cytology