bims-cateng 2023-09-10 papers

Issue of 2023–09–10
six papers selected by
Chance Bowman, Dartmouth College

Lab Chip. 2023 Sep 05.

Full-electric microfluidic platform to capture, analyze and selectively release single cells.

Ruben Van den Eeckhoudt, An-Sofie Christiaens, Frederik Ceyssens, Vasileios Vangalis, Kevin J Verstrepen, Nico Boon, Filip Tavernier, Michael Kraft, Irene Taurino.

Current single-cell technologies require large and expensive equipment, limiting their use to specialized labs. In this paper, we present for the first time a microfluidic device which demonstrates a combined method for full-electric cell capturing, analyzing, and selectively releasing with single-cell resolution. All functionalities are experimentally demonstrated on Saccharomyces cerevisiae. Our microfluidic platform consists of traps centered around a pair of individually accessible coplanar electrodes, positioned under a microfluidic channel. Using this device, we validate our novel Two-Voltage method for trapping single cells by positive dielectrophoresis (pDEP). Cells are attracted to the trap when a high voltage (VH) is applied. A low voltage (VL) holds the already trapped cell in place without attracting additional cells, allowing full control over the number of trapped cells. After trapping, the cells are analyzed by broadband electrochemical impedance spectroscopy. These measurements allow the detection of single cells and the extraction of cell parameters. Additionally, these measurements show a strong correlation between average phase change and cell size, enabling the use of our system for size measurements in biological applications. Finally, our device allows selectively releasing trapped cells by turning off the pDEP signal in their trap. The experimental results show the techniques potential as a full-electric single-cell analysis tool with potential for miniaturization and automation which opens new avenues towards small-scale, high throughput single-cell analysis and sorting lab-on-CMOS devices.

DOI: https://doi.org/10.1039/d3lc00645j

Mol Cell Proteomics. 2023 Sep 06. pii: S1535-9476(23)00154-8. [Epub ahead of print] 100643

A standardized and reproducible workflow for membrane glass slides in routine histology and spatial proteomics.

Thierry M Nordmann, Lisa Schweizer, Andreas Metousis, Marvin Thielert, Edwin Rodriguez, Lise Mette Rahbek-Gjerdrum, Pia-Charlotte Stadler, Michael Bzorek, Andreas Mund, Florian A Rosenberger, Matthias Mann.

Defining the molecular phenotype of single cells in-situ is key for understanding tissue architecture in health and disease. Advanced imaging platforms have recently been joined by spatial omics technologies, promising unparalleled insights into the molecular landscape of biological samples. Furthermore, high-precision laser microdissection of tissue on membrane glass slides is a powerful method for spatial omics technologies and single-cell type spatial proteomics in particular. However, current histology protocols have not been compatible with glass membrane slides and laser microdissection for automated staining platforms and routine histology procedures. This has prevented the combination of advanced staining procedures with laser microdissection. In this study we describe a novel method for handling glass membrane slides that enables automated, eight-color multiplexed immunofluorescence staining and high-quality imaging followed by precise laser-guided extraction of single cells. The key advance is the glycerol-based modification of heat-induced epitope retrieval protocols, termed 'G-HIER'. We find that this altered antigen-retrieval solution prevents membrane distortion. Importantly, G-HIER is fully compatible with current antigen retrieval workflows and mass spectrometry-based proteomics and does not affect proteome depth or quality. To demonstrate the versatility of G-HIER for spatial proteomics, we apply the recently introduced Deep Visual Proteomics technology to perform single-cell type analysis of adjacent suprabasal and basal keratinocytes of human skin. G-HIER overcomes previous incompatibility of standard and advanced staining protocols with membrane glass slides and enables robust integration with routine histology procedures, high-throughput multiplexed imaging and sophisticated downstream spatial omics technologies.

Keywords: Antigen retrieval; Deep Visual Proteomics; Glycerol; Histology; Laser microdissection; Membrane slides; Proteomics; Spatial proteomics

DOI: https://doi.org/10.1016/j.mcpro.2023.100643

Prog Mol Biol Transl Sci. 2023 ;pii: S1877-1173(23)00059-5. [Epub ahead of print]199 271-296

Universal and hypoimmunogenic pluripotent stem cells for clinical usage.

Tzu-Cheng Sung, Kailibinuer Maitiruze, Jiandong Pan, Jian Gong, Yongheng Bai, Xiaodong Pan, Akon Higuchi.

It is urgent to prepare and store large numbers of clinical trial grade human pluripotent stem (hPS) cells for off-the-shelf use in stem cell therapies. However, stem cell banks, which store off-the-shelf stem cells, need financial support and large amounts of technicians for daily cell maintenance. Therefore, it is valuable to create "universal" or "hypoimmunogenic" hPS cells with genome editing engineering by knocking in or out immune-related genes. Only a small number of universal or hypoimmunogenic hPS cell lines should be needed to store for off-the-shelf usage and reduce the large amounts of instruments, consumables and technicians. In this article, we consider how to create hypoimmunogenic or universal hPS cells as well as the demerits of the technology. β2-Microglobulin-knockout hPS cells did not harbor human leukocyte antigen (HLA)-expressing class I cells but led to the activation of natural killer cells. To escape the activities of macrophages and natural killer cells, homozygous hPS cells having a single allele of an HLA class I gene, such as HLA-C, were proposed. Major HLA class Ia molecules were knocked out, and CD47, HLA-G and PD-L1 were knocked in hPS cells utilizing CRISPR/Cas9 genome editing. Finally, some researchers are trying to generate universal hPS cells without genome editing. The cells evaded the activation of not only T cells but also macrophages and natural killer cells. These universal hPS cells have high potential for application in cell therapy.

Keywords: Cell therapy; Human leukocyte antigen; Hypoimmunogenic hPS cells; Immune privilege; Off-the-shelf cells; Universal hPS cells

DOI: https://doi.org/10.1016/bs.pmbts.2023.02.014

Front Bioeng Biotechnol. 2023 ;11 1255782

Laser-induced forward transfer based laser bioprinting in biomedical applications.

Jinlong Chang, Xuming Sun.

Bioprinting is an emerging field that utilizes 3D printing technology to fabricate intricate biological structures, including tissues and organs. Among the various promising bioprinting techniques, laser-induced forward transfer (LIFT) stands out by employing a laser to precisely transfer cells or bioinks onto a substrate, enabling the creation of complex 3D architectures with characteristics of high printing precision, enhanced cell viability, and excellent technical adaptability. This technology has found extensive applications in the production of biomolecular microarrays and biological structures, demonstrating significant potential in tissue engineering. This review briefly introduces the experimental setup, bioink ejection mechanisms, and parameters relevant to LIFT bioprinting. Furthermore, it presents a detailed summary of both conventional and cutting-edge applications of LIFT in fabricating biomolecule microarrays and various tissues, such as skin, blood vessels and bone. Additionally, the review addresses the existing challenges in this field and provides corresponding suggestions. By contributing to the ongoing development of this field, this review aims to inspire further research on the utilization of LIFT-based bioprinting in biomedical applications.

Keywords: 3D printing; biomedical applications; bioprinting; laser-induced forward transfer; tissue engineering

DOI: https://doi.org/10.3389/fbioe.2023.1255782

Int J Mol Sci. 2023 Sep 04. pii: 13655. [Epub ahead of print]24(17):

In Vitro Embryogenesis and Gastrulation Using Stem Cells in Mice and Humans.

Seung Yeon Oh, Seung Bin Na, Yoo Kyung Kang, Jeong Tae Do.

During early mammalian embryonic development, fertilized one-cell embryos develop into pre-implantation blastocysts and subsequently establish three germ layers through gastrulation during post-implantation development. In recent years, stem cells have emerged as a powerful tool to study embryogenesis and gastrulation without the need for eggs, allowing for the generation of embryo-like structures known as synthetic embryos or embryoids. These in vitro models closely resemble early embryos in terms of morphology and gene expression and provide a faithful recapitulation of early pre- and post-implantation embryonic development. Synthetic embryos can be generated through a combinatorial culture of three blastocyst-derived stem cell types, such as embryonic stem cells, trophoblast stem cells, and extraembryonic endoderm cells, or totipotent-like stem cells alone. This review provides an overview of the progress and various approaches in studying in vitro embryogenesis and gastrulation in mice and humans using stem cells. Furthermore, recent findings and breakthroughs in synthetic embryos and gastruloids are outlined. Despite ethical considerations, synthetic embryo models hold promise for understanding mammalian (including humans) embryonic development and have potential implications for regenerative medicine and developmental research.

Keywords: embryogenesis; gastrulation; stem cells; synthetic embryo

DOI: https://doi.org/10.3390/ijms241713655

J Math Biol. 2023 Sep 07. 87(4): 54

Adjusting the range of cell-cell communication enables fine-tuning of cell fate patterns from checkerboard to engulfing.

Simon Schardt, Sabine C Fischer.

During development, spatio-temporal patterns ranging from checkerboard to engulfing occur with precise proportions of the respective cell fates. Key developmental regulators are intracellular transcriptional interactions and intercellular signaling. We present an analytically tractable mathematical model based on signaling that reliably generates different cell type patterns with specified proportions. Employing statistical mechanics, We derived a cell fate decision model for two cell types. A detailed steady state analysis on the resulting dynamical system yielded necessary conditions to generate spatially heterogeneous patterns. This allows the cell type proportions to be controlled by a single model parameter. Cell-cell communication is realized by local and global signaling mechanisms. These result in different cell type patterns. A nearest neighbor signal yields checkerboard patterns. Increasing the signal dispersion, cell fate clusters and an engulfing pattern can be generated. Altogether, the presented model allows us to reliably generate heterogeneous cell type patterns of different kinds as well as desired proportions.

Keywords: Cell differentiation; Mathematical modeling; Pattern formation; Statistical mechanics

DOI: https://doi.org/10.1007/s00285-023-01959-9