CellCelector™ as a platform in isolating primary B cells for antibody discovery

Background The most robust strategy in antibody discovery is the use of immunized animals and the ability to isolate and immortalize immune B-cells to hybridoma for further interrogation. However, capturing the full repertoire of an immunized animal is labor intensive, time consuming, and limited in throughput. Therefore, techniques to directly mine the antibody repertoire of primary B-cells are of great importance in antibody discovery. Methods In the current study, we present a method to isolate individual antigen specific primary B-cells using the CellCellector™ single-cell isolation platform from XenoMouse® (XM) immunized with a recombinant therapeutic protein, EGFR. We screened a subset of CD138+ B-cells and identified 238 potential EGFR specific B-cells from 1,189 antibody secreting cells (ASCs) and isolated 94 by CellCellector. Results We identified a diverse set of heavy chain CDR sequences and cloned and expressed 20 into a standard human IgG1 antibody format. We further characterized and identified 13 recombinant antibodies that engage soluble and native forms of EGFR. By extrapolating the method to all 400,000 CD138+ B-cells extracted from one EGFR immunized XM, a potential 1,196 unique EGFR-specific antibodies could be discovered. Conclusions CellCelector allows for interrogating the B-cell pool directly and isolating B-cells specific to the therapeutic target of interest. Furthermore, antibody sequences recovered from isolated B-cells engage the native and recombinant target, demonstrating the CellCellector can serve as a platform in antibody discovery.

Advances in Single-Cell Printing

Single-cell analysis is becoming an indispensable tool in modern biological and medical research. Single-cell isolation is the key step for single-cell analysis. Single-cell printing shows several distinct advantages among the single-cell isolation techniques, such as precise deposition, high encapsulation efficiency, and easy recovery. Therefore, recent developments in single-cell printing have attracted extensive attention. We review herein the recently developed bioprinting strategies with single-cell resolution, with a special focus on inkjet-like single-cell printing. First, we discuss the common cell printing strategies and introduce several typical and advanced printing strategies. Then, we introduce several typical applications based on single-cell printing, from single-cell array screening and mass spectrometry-based single-cell analysis to three-dimensional tissue formation. In the last part, we discuss the pros and cons of the single-cell strategies and provide a brief outlook for single-cell printing.

Isolating and culturing of single microbial cells by laser ejection sorting technology

Single cell isolation and cultivation play an important role in studying physiology, gene expression and functions of microorganisms. A series of single-cell isolation technologies have been developed, among which single-cell ejection technology is one of the most promising. Single cell ejection technology has applied Laser Induced Forward Transfer Technique (LIFT) to isolate bacteria but the viability (or recovery rate) of cells after sorting has not been clarified in the current research progress. In this work, to keep the cells alive as much as possible, we propose a three-layer LIFT system (top layer: 25-nm aluminum film; second layer: 3 μm agar media; third layer: liquid containing bacterial) for the isolation and cultivation of single Gram-negative ( E. coli ), Gram-positive ( Lactobacillus rhamnosus GG, LGG), and eukaryotic microorganisms ( Saccharomyces cerevisiae ). The experiment results showed that the average survival rates for ejected pure single cells were 63% for Saccharomyces cerevisiae , 22% for E. coli DH5α, and 74% for LGG. In addition, we successfully isolated and cultured the GFP expressing E. coli JM109 from the mixture containing complex communities of soil bacteria by fluorescence signal. The average survival rate of E. coli JM109 was demonstrated to be 25.3%. In this study, the isolated and cultured single colonies were further confirmed by colony PCR and sequencing. Such precise sorting and cultivation technique of live single microbial cells could be coupled with other microscopic approaches to isolate single microorganisms with specific functions, revealing their roles in the natural community. Importance We developed a laser induced forward transfer (LIFT) technology to accurately isolate single live microbial cells. The cultivation recovery rates of the ejected single cells were 63% for Saccharomyces cerevisiae , 22% for E. coli DH5α, and 74% for Lactobacillus rhamnosus GG (LGG). Coupled LIFT with fluorescent microscope, we demonstrated that single cells of GFP expressing E. coli JM109 were sorted according to fluorescence signal from a complex community of soil bacteria, and subsequently cultured with 25% cultivation recovery rate. This single cell live sorting technology could isolate single microbes with specific functions, revealing their roles in the natural community.

Cross-reactivity of IgM anti-modified protein antibodies in rheumatoid arthritis despite limited mutational load

Background Anti-modified protein antibodies (AMPA) targeting citrullinated, acetylated and/or carbamylated self-antigens are hallmarks of rheumatoid arthritis (RA). Although AMPA-IgG cross-reactivity to multiple post-translational modifications (PTMs) is evident, it is unknown whether the first responding B cells, expressing IgM, display similar characteristics or if cross-reactivity is crucially dependent on somatic hypermutation (SHM). We now studied the reactivity of (germline) AMPA-IgM to further understand the breach of B cell tolerance and to identify if cross-reactivity depends on extensive SHM. Moreover, we investigated whether AMPA-IgM can efficiently recruit immune effector mechanisms. Methods Polyclonal AMPA-IgM were isolated from RA patients and assessed for cross-reactivity towards PTM antigens. AMPA-IgM B cell receptor sequences were obtained by single cell isolation using antigen-specific tetramers. Subsequently, pentameric monoclonal AMPA-IgM, their germline counterparts and monomeric IgG variants were generated. The antibodies were analysed on a panel of PTM antigens and tested for complement activation. Results Pentameric monoclonal and polyclonal AMPA-IgM displayed cross-reactivity to multiple antigens and different PTMs. PTM antigen recognition was still present, although reduced, after reverting the IgM into germline. Valency of AMPA-IgM was crucial for antigen recognition as PTM-reactivity significantly decreased when AMPA-IgM were expressed as IgG. Furthermore, AMPA-IgM was 15- to 30-fold more potent in complement-activation compared to AMPA-IgG. Conclusions We provide first evidence that AMPA-IgM are cross-reactive towards different PTMs, indicating that PTM (cross-)reactivity is not confined to IgG and does not necessarily depend on extensive somatic hypermutation. Moreover, our data indicate that a diverse set of PTM antigens could be involved in the initial tolerance breach in RA and suggest that AMPA-IgM can induce complement-activation and thereby inflammation.

Glyoxal as alternative fixative for single cell RNA sequencing

Single cell RNA sequencing (scRNA-seq) has become an important method to identify cell types, delineate the trajectories of cell differentiation in whole organisms and understand the heterogeneity in cellular responses. Nevertheless, sample collection and processing remain a severe bottleneck for scRNA-seq experiments. Cell isolation protocols often lead to significant changes in the transcriptomes of cells, requiring novel methods to preserve cell states. Here, we developed and benchmarked protocols using glyoxal as a fixative for scRNA-seq application. Using Drop-seq methodology, we detected high numbers of transcripts and genes from glyoxal-fixed Drosophila cells after scRNA-seq. The effective glyoxal fixation of transcriptomes in Drosophila and human cells was further supported by a high correlation of gene expression data between glyoxal-fixed and unfixed samples. Accordingly, we also found highly expressed genes overlapping to a large extent between experimental conditions. These results indicated that our fixation protocol did not induce considerable changes in gene expression and conserved the transcriptome for subsequent single cell isolation procedures. In conclusion, we present glyoxal as a suitable fixative for Drosophila cells and potentially cells of other species that allows high-quality scRNA-seq applications.