Isolation And Culture of Rat Intestinal Mucosal Microvascular Endothelial Cells Using Immunomagnetic Beads And Their Marker Expression

Microvascular endothelial cells (MVECs) have been an important tool in many research fields, while their purification remained challenging. This study aimed to establish a method for isolating rat intestinal mucosal MVECs using immunomagnetic beads, and characterize their proliferation ability and marker expression. Rat jejunum mucosas were scraped and digested by collagenase type II to obtain primary cell culture by the differential adhesion method. Magnetic beads were coated with anti-CD31 antibodies and incubated with the single-cell suspension of the primary cell culture. The CD31+ cells were separated using an automatic magnetic separation system, and their proliferation ability were assayed at different passages. The expression of factor VIII (FVIII), CD31, and CD34 was detected by immunofluorescence staining. Results indicated that rat intestinal mucosal MVECs grew into a contact-inhibited cobblestone-like monolayer after about 6 days, whose proliferation ability had no significant decline at passage 20. Three markers were generally expressed in the cells, and the fluorescence intensity of FVIII was higher after magnetic separation than before, and those of CD31 and CD34 remained similar. In conclusion, highly pure MVECs can be isolated from the primary cell culture of rat intestinal mucosas using magnetic beads coated with anti-CD31 antibodies, and magnetic separation may influence the expression of FVIII.

Inertial-Assisted Immunomagnetic Bioplatform towards Efficient Enrichment of Circulating Tumor Cells

Serving as an effective biomarker in liquid biopsy, circulating tumor cells (CTCs) can provide an accessible source for cancer biology study. For the in-depth evaluation of CTCs in cancer analysis, their efficient enrichment is essential, owing to their low abundance in peripheral blood. In this paper, self-assembled immunomagnetic beads were developed to isolate CTCs from the ordered bundles of cells under the assistance of the spiral inertial effect. Parametric numerical simulations were performed to explore the velocity distribution in the cross section. Based on this chip, rare CTCs could be recovered under the throughput of 500 μL/min, making this device a valuable supplement in cancer analysis, diagnostics, and therapeutics.

Development of IMBs-qPCR Detection Method for Yersinia enterocolitica Based on the foxA Gene

Yersinia enterocolitica is an important zoonotic pathogen, which seriously endangers food safety risk. In this study, the recombinant outer membrane protein OmpF and its antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Y. enterocolitica in food samples, combining the quantitative PCR detection with primers of virulence factor gene fox A for Yersinia enterocolitica contamination. The results showed that the capture efficiency of approximately 80% using anti-OmpF antibody-immunomagnetic beads and linearly dependent capture under 10 1 -10 5 CFU/mL Y. enterocolitica . compare with less than 10% capture of other bacteria. The detection limit of 64 CFU/mL was obtained by based on fox A gene PCR detection combined with capture of the anti-OmpF antibody-immunomagnetic beads to detect Yersinia enterocolitica in artificially contaminated milk and pork samples. Comparing with the culture method, the developed IMBs-qPCR method has higher consistency, less time consuming, which providing an effective alternative method for rapid detection of Y. enterocolitica in food.

Immunomagnetic sequential ultrafiltration (iSUF) platform for enrichment and purification of extracellular vesicles from biofluids

Extracellular vesicles (EVs) derived from tumor cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. However, current methods for EV isolation have limited specificity towards tumor-derived EVs that limit their clinical use. Here, we present an approach called immunomagnetic sequential ultrafiltration (iSUF) that consists of sequential stages of purification and enrichment of EVs in approximately 2 h. In iSUF, EVs present in different volumes of biofluids (0.5–100 mL) can be significantly enriched (up to 1000 times), with up to 99% removal of contaminating proteins (e.g., albumin). The EV recovery rate by iSUF for cell culture media (CCM), serum, and urine corresponded to 98.0% ± 3.6%, 96.0% ± 2.0% and 94.0% ± 1.9%, respectively (p > 0.05). The final step of iSUF enables the separation of tumor-specific EVs by incorporating immunomagnetic beads to target EV subpopulations. Serum from a cohort of clinical samples from metastatic breast cancer (BC) patients and healthy donors were processed by the iSUF platform and the isolated EVs from patients showed significantly higher expression levels of BC biomarkers (i.e., HER2, CD24, and miR21).