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

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

Research on CellSearch system's detection rate of cells and linearity performance detection method, so as to analyze the accurate and reasonable detection method to meet the CellSearch characteristics of the system.
Biomarkers, Tumor ; Cell Line, Tumor ; Cell Separation ; methods ; Humans ; Neoplastic Cells, Circulating

The death of patients with gastric cancer is mainly due to its recurrence and metastasis, and circulating tumor cell (CTC) is the necessary condition of metastasis. As liquid biopsy, CTC detection has its certain clinical significance. The detection is required after enrichment because circulating tumor cells are rare. Many enrichment methods have been developed: methods based on physical characteristics of TCT, like density, size and dielectric properties and so on; immunogenicity, like Cell Search System; and microfluidic chip technology. The immunofluorescence is commonly used to identify CTC in gastric cancer and the isolated CTC can also be used for the following analysis on the level of nucleic acid, protein and gene regulation. Detection of CTC in gastric cancer is helpful to judge the prognosis, assess staging, monitor the curative effect and guide the development of drug. There are many challenges for clinical transformation of CTC: the lower enrichment efficiency, the less specific surface markers, the uncertain diagnostic efficiency and so on, but it also has the good research prospect because it is non-invasive, repeatable and can real-time monitor the condition and guide the clinical treatment compared with pathological biopsy. In this paper, the detection and identification methods, and clinical value of CTC in gastric cancer patients are reviewed.
Biomarkers, Tumor ; Biopsy ; Cell Separation ; methods ; Cytodiagnosis ; methods ; Flow Cytometry ; methods ; Fluorescent Antibody Technique ; methods ; Humans ; Microchip Analytical Procedures ; methods ; Neoplasm Recurrence, Local ; prevention & control ; Neoplasm Staging ; methods ; Neoplastic Cells, Circulating ; metabolism ; pathology ; Prognosis ; Secondary Prevention ; Stomach Neoplasms ; blood ; diagnosis ; genetics ; therapy ; Treatment Outcome

Adipose-derived stem cells (ADSCs) are an attractive source of material for mesenchymal stem cell research due to the abundance of adipose and relative ease of access compared with bone marrow. A key consideration for research is whether cell isolation methods can be improved, to reduce the process steps needed to isolate and expand cell material. In the current study, we used macroporous biopolymer microcarriers to isolate primary ADSCs. We found that the method was capable of isolating ADSCs that were subsequently capable of being transferred to culture dishes and expanded in vitro. Moreover, flow cytometry revealed that they expressed typical stem cell markers and were capable of undergoing tri-lineage differentiation. In summary, it is feasible to use biopolymer microcarriers for retrieval of viable ADSCs that retain identity markers of stem cell function.
Adult Stem Cells ; Animals ; Biopolymers* ; Bone Marrow ; Cell Separation ; Flow Cytometry ; In Vitro Techniques ; Mesenchymal Stromal Cells ; Methods ; Rats* ; Stem Cells*

Mesenchymal stem cell (MSC)-mediated therapy has been shown to be clinically effective in regenerating tissue defects. For improved regenerative therapy, it is critical to isolate homogenous populations of MSCs with high capacity to differentiate into appropriate tissues. The utilization of stem cell surface antigens provides a means to identify MSCs from various tissues. However, few surface markers that consistently isolate highly regenerative MSCs have been validated, making it challenging for routine clinical applications and making it all the more imperative to identify reliable surface markers. In this study, we used three surface marker combinations: CD51/CD140α, CD271, and STRO-1/CD146 for the isolation of homogenous populations of dental mesenchymal stem cells (DMSCs) from heterogeneous periodontal ligament cells (PDLCs). Fluorescence-activated cell sorting analysis revealed that 24% of PDLCs were CD51(+)/CD140α(+), 0.8% were CD271(+), and 2.4% were STRO-1(+)/CD146(+). Sorted cell populations were further assessed for their multipotent properties by inducing osteogenic and chondrogenic differentiation. All three subsets of isolated DMSCs exhibited differentiation capacity into osteogenic and chondrogenic lineages but with varying degrees. CD271(+) DMSCs demonstrated the greatest osteogenic potential with strong induction of osteogenic markers such as DLX5, RUNX2, and BGLAP. Our study provides evidence that surface marker combinations used in this study are sufficient markers for the isolation of DMSCs from PDLCs. These results provide important insight into using specific surface markers for identifying homogenous populations of DMSCs for their improved utilization in regenerative medicine.
Adaptor Proteins, Signal Transducing ; analysis ; Adult ; Aggrecans ; analysis ; Antigens, CD ; analysis ; Antigens, Surface ; analysis ; CD146 Antigen ; analysis ; Cell Differentiation ; physiology ; Cell Lineage ; Cell Separation ; methods ; Cells, Cultured ; Chondrogenesis ; physiology ; Collagen Type II ; analysis ; Core Binding Factor Alpha 1 Subunit ; analysis ; Flow Cytometry ; methods ; Homeodomain Proteins ; analysis ; Humans ; Integrin alphaV ; analysis ; Mesenchymal Stromal Cells ; cytology ; physiology ; Multipotent Stem Cells ; cytology ; physiology ; Nerve Tissue Proteins ; analysis ; Osteogenesis ; physiology ; Periodontal Ligament ; cytology ; Receptor, Platelet-Derived Growth Factor alpha ; analysis ; Receptors, Nerve Growth Factor ; analysis ; SOX9 Transcription Factor ; analysis ; Time Factors ; Transcription Factors ; analysis

Mesenchymal stem cells (MSCs) are a promising tool in regenerative medicine due to their capacity to differentiate into multiple lineages. In addition to MSCs isolated from bone marrow (BMSCs), adult MSCs are isolated from craniofacial tissues including dental pulp tissues (DPs) using various stem cell surface markers. However, there has been a lack of consensus on a set of surface makers that are reproducibly effective at isolating putative multipotent dental mesenchymal stem cells (DMSCs). In this study, we used different combinations of surface markers (CD51/CD140α, CD271, and STRO-1/CD146) to isolate homogeneous populations of DMSCs from heterogeneous dental pulp cells (DPCs) obtained from DP and compared their capacity to undergo multilineage differentiation. Fluorescence-activated cell sorting revealed that 27.3% of DPCs were CD51(+)/CD140α(+), 10.6% were CD271(+), and 0.3% were STRO-1(+)/CD146(+). Under odontogenic conditions, all three subsets of isolated DMSCs exhibited differentiation capacity into odontogenic lineages. Among these isolated subsets of DMSCs, CD271(+) DMSCs demonstrated the greatest odontogenic potential. While all three combinations of surface markers in this study successfully isolated DMSCs from DPCs, the single CD271 marker presents the most effective stem cell surface marker for identification of DMSCs with high odontogenic potential. Isolated CD271(+) DMSCs could potentially be utilized for future clinical applications in dentistry and regenerative medicine.
Adult ; Adult Stem Cells ; cytology ; Antigens, CD ; analysis ; Antigens, Surface ; analysis ; Biomarkers ; analysis ; CD146 Antigen ; analysis ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cell Lineage ; Cell Separation ; methods ; Cells, Cultured ; Chondrogenesis ; physiology ; Dental Pulp ; cytology ; Flow Cytometry ; methods ; Humans ; Integrin alphaV ; analysis ; Mesenchymal Stromal Cells ; cytology ; Multipotent Stem Cells ; cytology ; Nerve Tissue Proteins ; analysis ; Odontogenesis ; physiology ; Receptor, Platelet-Derived Growth Factor alpha ; analysis ; Receptors, Nerve Growth Factor ; analysis

Recently, gingival margin-derived stem/progenitor cells isolated via STRO-1/magnetic activated cell sorting (MACS) showed remarkable periodontal regenerative potential in vivo. As a second-stage investigation, the present study's aim was to perform in vitro characterisation and comparison of the stem/progenitor cell characteristics of sorted STRO-1-positive (MACS⁺) and STRO-1-negative (MACS⁻) cell populations from the human free gingival margin. Cells were isolated from the free gingiva using a minimally invasive technique and were magnetically sorted using anti-STRO-1 antibodies. Subsequently, the MACS⁺ and MACS⁻ cell fractions were characterized by flow cytometry for expression of CD14, CD34, CD45, CD73, CD90, CD105, CD146/MUC18 and STRO-1. Colony-forming unit (CFU) and multilineage differentiation potential were assayed for both cell fractions. Mineralisation marker expression was examined using real-time polymerase chain reaction (PCR). MACS⁺ and MACS(-) cell fractions showed plastic adherence. MACS⁺ cells, in contrast to MACS⁻ cells, showed all of the predefined mesenchymal stem/progenitor cell characteristics and a significantly higher number of CFUs (P<0.01). More than 95% of MACS⁺ cells expressed CD105, CD90 and CD73; lacked the haematopoietic markers CD45, CD34 and CD14, and expressed STRO-1 and CD146/MUC18. MACS⁻ cells showed a different surface marker expression profile, with almost no expression of CD14 or STRO-1, and more than 95% of these cells expressed CD73, CD90 and CD146/MUC18, as well as the haematopoietic markers CD34 and CD45 and CD105. MACS⁺ cells could be differentiated along osteoblastic, adipocytic and chondroblastic lineages. In contrast, MACS⁻ cells demonstrated slight osteogenic potential. Unstimulated MACS⁺ cells showed significantly higher expression of collagen I (P<0.05) and collagen III (P<0.01), whereas MACS⁻ cells demonstrated higher expression of osteonectin (P<0.05; Mann-Whitney). The present study is the first to compare gingival MACS⁺ and MACS⁻ cell populations demonstrating that MACS⁺ cells, in contrast to MACS⁻ cells, harbour stem/progenitor cell characteristics. This study also validates the effectiveness of the STRO-1/MACS⁺ technique for the isolation of gingival stem/progenitor cells. Human free gingival margin-derived STRO-1/MACS⁺ cells are a unique renewable source of multipotent stem/progenitor cells.
Base Sequence ; Cell Differentiation ; Cell Lineage ; Cells, Cultured ; DNA Primers ; Gene Expression Profiling ; Gingiva ; cytology ; metabolism ; Humans ; Immunomagnetic Separation ; methods ; Immunophenotyping ; Real-Time Polymerase Chain Reaction

OBJECTIVETo isolate, identify and culture human spermatogonial stem cells (SSC) and then obtain purified and enriched human SSCs for research and application.

METHODSWe detected the expression of CD90 in the human testis using the immunofluorescence technique and isolated human testicular spermatogenic cells by two-step enzymatic digestion, followed by differential plating and magnetic-activated cell sorting (MACS) with CD90 as an SSC marker. Then we identified the isolated CD90-positive spermatogenic cells by RT-PCR and immunocytochemistry, and meanwhile cocultured them with Sertoli cells in SG medium in vitro.

RESULTSThe isolated CD90-positive cells showed a relatively homogeneous characteristic in size and morphology and expressed the genes specific for human SSCs, with high expressions (90.5%) of GFRA1, GPR125, and UCHL1. After coculture with Sertoli cells in the SG medium for 2 weeks, the isolated CD90-positive cells maintained a good activity.

CONCLUSIONCD90 can be regarded as a speci- fic marker for human SSCs and used to obtain highly enriched human SSCs by differential plating and MACS. Furthermore, the isolated human SSCs can be cultured in SG medium in vitro.

Adult Stem Cells ; cytology ; Biomarkers ; metabolism ; Cell Separation ; methods ; Cell Shape ; Cell Size ; Coculture Techniques ; Glial Cell Line-Derived Neurotrophic Factor Receptors ; metabolism ; Humans ; Immunohistochemistry ; Male ; Receptors, G-Protein-Coupled ; metabolism ; Sertoli Cells ; Spermatogonia ; cytology ; Testis ; metabolism ; Thy-1 Antigens ; isolation & purification ; metabolism ; Ubiquitin Thiolesterase ; metabolism