Ultrahigh-throughput magnetic sorting of large blood volumes for epitope-agnostic isolation of circulating tumor cells.

Circulating tumor cell (CTC)-based liquid biopsies provide unique opportunities for cancer diagnostics, treatment selection, and response monitoring, but even with advanced microfluidic technologies for rare cell detection the very low number of CTCs in standard 10-mL peripheral blood samples limits their clinical utility. Clinical leukapheresis can concentrate mononuclear cells from almost the entire blood volume, but such large numbers and concentrations of cells are incompatible with current rare cell enrichment technologies. Here, we describe an ultrahigh-throughput microfluidic chip, LPCTC-iChip, that rapidly sorts through an entire leukapheresis product of over 6 billion nucleated cells, increasing CTC isolation capacity by two orders of magnitude (86% recovery with 105 enrichment). Using soft iron-filled channels to act as magnetic microlenses, we intensify the field gradient within sorting channels. Increasing magnetic fields applied to inertially focused streams of cells effectively deplete massive numbers of magnetically labeled leukocytes within microfluidic channels. The negative depletion of antibody-tagged leukocytes enables isolation of potentially viable CTCs without bias for expression of specific tumor epitopes, making this platform applicable to all solid tumors. Thus, the initial enrichment by routine leukapheresis of mononuclear cells from very large blood volumes, followed by rapid flow, high-gradient magnetic sorting of untagged CTCs, provides a technology for noninvasive isolation of cancer cells in sufficient numbers for multiple clinical and experimental applications.

Magnetic Suspension Array Technology: Controlled Synthesis and Screening in Microfluidic Networks.

Information tagging and processing are vital in information-intensive applications, e.g., telecommunication and high-throughput drug screening. Magnetic suspension array technology may offer intrinsic advantages to screening applications by enabling high distinguishability, the ease of code generation, and the feasibility of fast code readout, though the practical applicability of magnetic suspension array technology remains hampered by the lack of quality administration of encoded microcarriers. Here, a logic-controlled microfluidic system enabling controlled synthesis of magnetic suspension arrays in multiphase flow networks is realized. The smart and compact system offers a practical solution for the quality administration and screening of encoded magnetic microcarriers and addresses the universal need of process control for synthesis in microfluidic networks, i.e., on-demand creation of droplet templates for high information capacity. The demonstration of magnetic suspension array technology enabled by magnetic in-flow cytometry opens the avenue toward point-of-care multiplexed bead-based assays, clinical diagnostics, and drug discovery.

Isolation and Culture Techniques for Fetal Mouse Germ Cells.

The fetal gonad contains a great variety of differentiating cell populations, of which germ cells make up a relatively small percentage. In order to study germ cell-specific gene and protein expression, as well as determine direct effects of signaling molecules, it is necessary to prepare enriched populations of germ cells and maintain them in culture for several hours to multiple days. The protocols in this chapter are designed to provide a guide for the isolation or enrichment of primordial germ cells (from 9.5 days post coitum (dpc) to 18.5 dpc) by flow cytometry (Subheading 3.1) or magnetic sorting (Subheading 3.2), followed by feeder-free primary germ cell culture (Subheading 3.3).

Cancer Cell Targeting, Magnetic Sorting, and SERS Detection through Cell Surface Receptors.

Integrins are cellular surface receptors responsible for the activation of many cellular pathways in cancer. These integrin proteins can be specifically targeted by small peptide sequences that offer the potential for the differentiation of cellular subpopulations by using magnetically assisted cellular sorting techniques. By adding a gold shell to the magnetic nanoparticles, these integrin-peptide interactions can be differentiated by surface-enhanced Raman spectroscopy (SERS), providing a quick and reliable method for on-target binding. In this paper, we demonstrate the ability to differentiate the peptide-protein interactions of the small peptides CDPGYIGSR and cyclic RGDfC functionalized on gold-coated magnetic nanoparticles with the integrins they are known to bind to using their SERS signal. SW480 and SW620 colorectal cancer cells known to have the integrins of interest were then magnetically sorted using these functionalized nanoparticles, suggesting differentiation between the sorted populations and integrin populations among the two cell lines.

Negative Magnetic Sorting Preserves the Functionality of Ex Vivo Cultivated Non-Adherent Human Monocytes.

BACKGROUND: Monocyte-derived macrophages or dendritic cells are of increasing interest for cellular therapeutic products to treat inflammation-related diseases and cancer. However, the isolation method and the culture conditions applied influence the functionality of cells. For some approaches, the adhesion-induced differentiation into macrophages must be prevented to maintain functions attributed to circulating monocytes. The effects of the isolation method on the functionality of non-adherent peripheral monocytes have not yet been investigated. METHODS: The present study examines the impact of the isolation method on cell viability, growth, metabolism, inflammation-induced cytokine response, migratory capacity, and adherence of non-adherent human peripheral monocytes. The monocytes were isolated by magnetic sorting using either positive or negative selection and cultured in cell-repellent plates. RESULTS: The purity and yield of monocytes were higher after positive selection. However, the adherence and migratory capacity, cytokine response, and metabolic activity were decreased compared to negatively selected monocytes. The impaired functionality presented in combination with cell shrinking, thus, indicates the start of cell viability loss. Negatively selected non-adherent monocytes showed no impairment in functionality, and the viability remained high. In conclusion, this approach is better suited for conducting ex vivo modifications of monocytes prior to the intended experimental setup or therapeutic application.

Impact of CD138 Magnetic Bead-based Positive Selection on Bone Marrow Plasma Cell Surface Markers.

BACKGROUND: Isolation of malignant plasma cells from bone marrow of patients with monoclonal gammopathies is critical for studies into the disease biology. The plasma cells are typically isolated by positive selection using plasma cell markers such as CD138. Here we have examined the effect of CD138 magnetic bead selection on the expression of other surface phenotypic markers on plasma cells. MATERIALS AND METHODS: Bone marrow aspirates from 16 patients were split and prepared using 2 methods before staining for flow cytometric evaluation. The first method (whole bone marrow) used an ammonium-chloride-potassium lyse of whole bone marrow followed by 2 phosphate buffered saline washes. The second method used CD138-positive magnetic sorting technology (Stem Cell Technology). The cells were run on the FACSCanto flow cytometer after staining for CD38, CD45, CD56, activation markers CD71, CD69, CD154, adhesion markers CD49d, CD49e, CD11a, CD11b, and CD66, B cell markers CD19 and CD20, and for clonality. RESULTS: There was a substantial loss in the expression of CD71, CD11b, CD11a, CD69, and CD49e on plasma cells following CD138-based sorting. Moreover, in 8 of the 16 cases, there was a nearly complete loss of the CD45-positive subset with a loss of discrimination between CD45-negative and CD45-positive plasma cell subsets in the remaining CD138-sorted preparations. CONCLUSIONS: The change in immunophenotype of the plasma cells on magnetic sorting should be kept in mind when isolating plasma cells using CD138-positive selection for analysis of plasma cells. The technique for characterizing plasma cells should be selected based on the study design to prevent loss of crucial and valuable information.

A Lab-On-chip Tool for Rapid, Quantitative, and Stage-selective Diagnosis of Malaria.

Malaria remains the most important mosquito-borne infectious disease worldwide, with 229 million new cases and 409.000 deaths in 2019. The infection is caused by a protozoan parasite which attacks red blood cells by feeding on hemoglobin and transforming it into hemozoin. Despite the WHO recommendation of prompt malaria diagnosis, the quality of microscopy-based diagnosis is frequently inadequate while rapid diagnostic tests based on antigens are not quantitative and still affected by non-negligible false negative/positive results. PCR-based methods are highly performant but still not widely used in endemic areas. Here, a diagnostic tool (TMek), based on the paramagnetic properties of hemozoin nanocrystals in infected red blood cells (i-RBCs), is reported on. Exploiting the competition between gravity and magnetic forces, i-RBCs in a whole blood specimen are sorted and electrically detected in a microchip. The amplitude and time evolution of the electrical signal allow for the quantification of i-RBCs (in the range 10-105 i-RBC µL-1) and the distinction of the infection stage. A preliminary validation study on 75 patients with clinical suspect of malaria shows on-field operability, without false negative and a few false positive results. These findings indicate the potential of TMek as a quantitative, stage-selective, rapid test for malaria.

Isolation and characterization of immune cells from the tumor microenvironment of genetically engineered pediatric high-grade glioma models using the sleeping beauty transposon system.

Gliomas are the most common malignant brain tumors in the pediatric population. Even though great efforts have been made to understand their distinctive molecular characteristics, there has not been any improvements in the median survival in decades. In children, high-grade glial tumors have a median survival of 9-15 months. It has recently been demonstrated that pediatric high-grade gliomas (pHGG) are biologically and molecularly different from the adult counterparts, which could explain why conventional treatments universally fail. The development of an in vivo pHGG model harboring the specific genetic alterations encountered in pediatric gliomas is imperative in order to study the molecular basis that drives the progression and aggressiveness of these tumors. It would also enable harnessing these results for the development of novel therapeutic approaches. Our lab has implemented a method to induce brain tumors using transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mouse brain. One of the main advantages of this method is that tumors are induced by altering the genome of the host cells, allowing us to recapitulate the salient features of the human disease. In this chapter we describe a method to isolate two cell populations from tumors generated in situ in mice, i.e., one population enriched in tumor cells and another population enriched in CD45+ cells. We also present methodologies as to how tumor infiltrating immune cells can be phenotypically characterized using flow cytometry.

A Protocol for Non-biased Identification of RNAs Transferred Between Heterologous Mammalian Cell Types Using RNA Tagging, Cell Sorting, and Sequencing.

Intercellular communication is a major hallmark of multicellular organisms and is responsible for coordinating cell and tissue differentiation, immune responses, synaptic transmission, and both paracrine and endocrine signaling, for example. Small molecules, peptides, and proteins have all been studied extensively as mediators of intercellular communication; however, RNAs have also been shown recently to transfer between cells. In mammalian cells, microRNAs, tRNAs, short noncoding RNAs, mRNA fragments, as well as full-length mRNAs have all been shown to transfer between cells either by exosomes or by membrane nanotubes. We have previously described nanotube-mediated cell-cell transfer of specific mRNAs between heterologous mammalian cell types cultured in vitro. Here, we describe a simple method for the unbiased and quantitative identification of the complete range of transferred mRNAs (i.e., the mRNA transferome) in one population of mammalian cells following co-culture with another population. After co-culture, the individual cell populations are sorted by magnetic bead-mediated cell sorting and the transferred RNAs are then identified by downstream analysis methods, such as RNA sequencing. Application of this technique not only allows for determination of the mRNA transferome, but can also reveal changes in the native transcriptome of a cell population after co-culture. This can indicate the effect that co-culture and intercellular transfer of mRNA have upon cell physiology.

Isolation of Epidermal Progenitor Cells from Rat Tympanic Membrane.

The eardrum is an important structural component for hearing, but it is delicate and subject to traumatic injury and disease. Healing mechanisms are activated after injury but sometimes healing fails and chronic perforations develop, requiring surgical intervention. To model the wound healing responses we established a simple method for isolating keratinocytes and progenitors from individual eardrums. The central region of the eardrum contains epidermal proliferative centers that produce keratinocytes which migrate to cover the eardrum surface. We dissected out the central region and explanted it to the plastic membrane of a culture well insert. Epidermal cells grew from the explant onto the surface of the insert membrane. The cells could be serially harvested and passaged for continuous culture and characterization. Magnetic immunoseparation methods were used to enrich for epithelial cells with stem cell-like characteristics. Proliferation and migration in vitro was demonstrated, and the cells were shown suitable for tissue engineering applications.

Selection of Antibody Fragments by Yeast Display.

The critical need for renewable, high-quality affinity reagents in biological research, as well as for diagnostic and therapeutic applications, has required the development of new platforms of discovery. Yeast display is one of the main methods of in vitro display technology with phage display. Yeast display has been chosen by numerous groups to refine both affinity and specificity of antibodies because it enables fine discrimination between mutant clones of similar affinity. In addition, the construction of display libraries of antibody fragments in yeast permits to sample the immune antibody repertoire more fully than using phage. This chapter gives an updated overview of the available systems of yeast display platforms and libraries, followed up by technical descriptions of selection methods of antibody fragments by yeast display.

Mouse Fetal Germ Cell Isolation and Culture Techniques.

The fetal gonad contains a great variety of differentiating cell populations, of which germ cells make up a small percentage. In order to study germ cell-specific gene and protein expression, as well as determine direct effects of signaling molecules, it is necessary to prepare enriched populations of germ cells and maintain them in culture for several hours to multiple days. The protocols in this chapter are designed to provide a guide for the isolation or enrichment of mouse primordial germ cells (from 9.5 days postcoitum (dpc) to 18.5 dpc) by flow cytometry (Subheading 3.1) or magnetic sorting (Subheading 3.2), followed by primary germ cell culture (Subheading 3.3).

A type of novel fluorescent magnetic carbon quantum dots for cells imaging and detection.

A new type of multifunctional fluorescent magnetic carbon quantum dots SPIO@CQDs(n) ([superparamagnetic iron oxide nanoparticles (SPIO), carbon quantum dots, (CQDs)]) with magnetic and fluorescence properties was designed and prepared through layer-by-layer self-assembly method. The as-synthesized SPIO@CQDs(n) exhibited different emission colors including blue, green, and red when they were excited at different excitation wavelengths, and its fluorescent intensity increased as the increase of CQD layer (n). SPIO@CQDs(n) with quite low toxicity could mark cytoplasm with fluorescence by means of nonimmune markers. The mixture sample of liver cells L02 and hepatoma carcinoma cells HepG2 was taken as an example, and HepG2 cells were successfully separated and detected effectively by SPIO@CQDs(n), with a separation rate of 90.31%. Importantly, the designed and prepared SPIO@CQDs( n ) are certified to be wonderful biological imaging and magnetic separation regents.