Systematic Study of Single-Cell Isolation from Musculoskeletal Tissues for Single-Cell Sequencing

Abstract Background: The single-cell platform provided revolutionary way to study cellular biology. Technologically, a sophistic protocol of isolating qualified single cells would be key to deliver to single-cell platform, which requires high cell viability, high cell yield and low content of cell aggregates or doublets. For musculoskeletal tissues, like bone, cartilage, nucleus pulposus, tendons, etc. as well as their pathological state, which are tense and dense, it’s full of challenge to efficiently and rapidly prepare qualified single-cell suspension. Conventionally, enzymatic dissociation methods were wildly used but lack of quality control. In the present study, we designed specific enzymatic treatment protocols for several human pathological musculoskeletal tissues, including degenerated nucleus pulposus, ossifying posterior longitudinal ligament and knee articular cartilage with osteoarthritis, aiming to rapidly and efficiently harvest qualified single-cell suspensions to meet the requirements of single-cell RNA-sequencing (scRNA-seq).Results: The single-cell suspensions from human degenerated nucleus pulposus and ossifying posterior longitudinal ligaments were both qualified after systematic quality control. Bioanalyzer trace showed expected cDNA size distribution of the scRNA-seq library. A clear separation of cellular barcodes from background partitions were verified by the barcode-rank plot after sequencing. However, we failed to obtain eligible samples from articular cartilage due to low cell viability and excessive cell aggregates and doublets. Conclusions: In conclusion, we provided rapid and efficient single-cell isolation protocols for human degenerated nucleus pulposus and ossifying posterior longitudinal ligament, which could be applied for scRNA-seq. More efforts will be made on improving the protocols for human articular cartilage.

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Development of a Multi-Arm Bioprinter for Hybrid Tissue Engineering

Volume 1: Processing ◽

10.1115/msec2013-1025 ◽

2013 ◽

Cited By ~ 1

Author(s):

Howard Chen ◽

Ibrahim T. Ozbolat

Keyword(s):

Tissue Engineering ◽

Articular Cartilage ◽

Cell Viability ◽

Progenitor Cells ◽

Layer Structure ◽

Single Layer ◽

Cartilage Tissue ◽

High Cell ◽

Cell Spheroids ◽

Multiple Materials

This paper highlights the development of a multi-arm bioprinter (MABP) capable of concurrent deposition of multiple materials with independent dispensing parameters including deposition speed, material dispensing rate and frequency for functional zonal-stratified articular cartilage tissue fabrication. The MABP consists of two Cartesian robots mounted in parallel on the same mechanical frame. This platform is used for concurrent filament fabrication and cell spheroid deposition. A single-layer structure is fabricated and concurrently deposited with spheroids to validate this system. Preliminary results showed that the MABP was able to produce filaments and spheroids with well-defined geometry and high cell viability. The resulting filament width has a variation of +/-170 μm and the center-to-center filament distance was within 100 μm of the specified distance. This fabrication system is aimed to be further refined for printing structures with varying porosities to mimic the natural cartilage structure in order to produce functional tissue-engineered articular cartilage using cell spheroids containing cartilage progenitor cells (CPCs).

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Nano-localized single-cell nano-electroporation

Lab on a Chip ◽

10.1039/d0lc00712a ◽

2020 ◽

Vol 20 (22) ◽

pp. 4194-4204

Author(s):

Tuhin Subhra Santra ◽

Srabani Kar ◽

Hwan-You Chang ◽

Fan-Gang Tseng

Keyword(s):

Cell Viability ◽

Single Cell ◽

Intracellular Delivery ◽

High Cell ◽

Highly Efficient

We demonstrated nano-electroporation technique to create transient nano-holes at single or multiple nano-localized positions of a single-cell for a highly efficient intracellular delivery with high cell viability.

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Nanolocalized Single-Cell-Membrane Nanoelectroporation: For higher efficiency with high cell viability.

IEEE Nanotechnology Magazine ◽

10.1109/mnano.2014.2312031 ◽

2014 ◽

Vol 8 (1) ◽

pp. 30-34 ◽

Cited By ~ 7

Author(s):

Tuhin Subhra Santra ◽

Srabani Kar ◽

Jayant Borana ◽

Pen-Cheng Wang ◽

Fan-Gang Tseng

Keyword(s):

Cell Membrane ◽

Cell Viability ◽

Single Cell ◽

High Cell

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Impedance-Based Single-Cell Pipetting

SLAS TECHNOLOGY Translating Life Sciences Innovation ◽

10.1177/2472630320911636 ◽

2020 ◽

Vol 25 (3) ◽

pp. 222-233

Author(s):

David Bonzon ◽

Georges Muller ◽

Jean-Baptiste Bureau ◽

Nicolas Uffer ◽

Nicolas Beuchat ◽

...

Keyword(s):

Cell Viability ◽

Single Cell ◽

Gold Standard ◽

Single Cells ◽

Cell Isolation ◽

Design Parameters ◽

Cell Line Development ◽

A Cell ◽

Single Cell Isolation ◽

Biological Methods

Many biological methods are based on single-cell isolation. In single-cell line development, the gold standard involves the dilution of cells by means of a pipet. This process is time-consuming as it is repeated over several weeks to ensure clonality. Here, we report the modeling, designing, and testing of a disposable pipet tip integrating a cell sensor based on the Coulter principle. We investigate, test, and discuss the effects of design parameters on the sensor performances with an analytical model. We also describe a system that enables the dispensing of single cells using an instrumented pipet coupled with the sensing tip. Most importantly, this system allows the recording of an impedance trace to be used as proof of single-cell isolation. We assess the performances of the system with beads and cells. Finally, we show that the electrical detection has no effect on cell viability.

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Quality control of direct cell–mineral adhesion measurements in air and liquid using inverse AFM imaging

RSC Advances ◽

10.1039/d1ra00110h ◽

2021 ◽

Vol 11 (10) ◽

pp. 5384-5392

Author(s):

Abd Alaziz Abu Quba ◽

Gabriele E. Schaumann ◽

Mariam Karagulyan ◽

Doerte Diehl

Keyword(s):

Quality Control ◽

Single Cell ◽

Single Cell Level ◽

Cell Level ◽

C Cell ◽

Afm Imaging ◽

Direct Cell ◽

Mineral Interaction

Setup for a reliable cell-mineral interaction at the single-cell level, (a) study of the mineral by a sharp tip, (b) study of the bacterial modified probe by a characterizer, (c) cell-mineral interaction, (d) subsequent check of the modified probe.

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A microfluidic device enabling deterministic single cell trapping and release

Lab on a Chip ◽

10.1039/d1lc00302j ◽

2021 ◽

Author(s):

Huichao Chai ◽

Yongxiang Feng ◽

Fei Liang ◽

Wenhui Wang

Keyword(s):

Chemical Analysis ◽

Single Cell ◽

Microfluidic Device ◽

Single Cells ◽

Cell Isolation ◽

Cell Trapping ◽

Analysis Techniques ◽

Single Cell Trapping ◽

Single Cell Isolation ◽

Made In

Successful single-cell isolation is a pivotal technique for subsequent biological and chemical analysis of single cells. Although significant advances have been made in single-cell isolation and analysis techniques, most passive...

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Optimization of Spheroid Colony Culture and Cryopreservation of Nucleus Pulposus Cells for the Development of Intervertebral Disc Regenerative Therapeutics

Applied Sciences ◽

10.3390/app11083309 ◽

2021 ◽

Vol 11 (8) ◽

pp. 3309

Author(s):

Kosuke Sako ◽

Daisuke Sakai ◽

Yoshihiko Nakamura ◽

Erika Matsushita ◽

Jordy Schol ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Viability ◽

Nucleus Pulposus ◽

Mass Culture ◽

Formation Rate ◽

Proliferation Rate ◽

Cell Phenotype ◽

Mrna Levels ◽

Spheroid Formation ◽

Positive Nucleus

After the discovery of functionally superior Tie2-positive nucleus pulposus (NP) progenitor cells, new methods were needed to enable mass culture and cryopreservation to maintain these cells in an undifferentiated state with high cell yield. We used six types of EZSPHERE® dishes, which support spheroid-forming colony culture, and examined NP cell spheroid-formation ability, number, proliferation, and mRNA expression of ACAN, COL1A2, COL2A1, and ANGPT1. Six different types of cryopreservation solutions were examined for potential use in clinical cryopreservation by comparing the effects of exposure time during cryopreservation on cell viability, Tie2-positivity, and cell proliferation rates. The spheroid formation rate was 45.1% and the cell proliferation rate was 7.75 times using EZSPHERE® dishes. The mRNA levels for COL2A1 and ANGPT1 were also high. In cryopreservation, CryoStor10 (CS10) produced ≥90% cell viability and a high proliferation rate after thawing. CS10 had a high Tie2-positive rate of 12.6% after culturing for 5 days after thawing. These results suggest that EZSPHERE enabled colony formation in cell culture without the use of hydrogel products and that CS10 is the best cryopreservation medium for retaining the NP progenitor cell phenotype and viability. Together, these data provide useful information of NP cell-based therapeutics to the clinic.

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Sparsely-connected autoencoder (SCA) for single cell RNAseq data mining

npj Systems Biology and Applications ◽

10.1038/s41540-020-00162-6 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Luca Alessandri ◽

Francesca Cordero ◽

Marco Beccuti ◽

Nicola Licheri ◽

Maddalena Arigoni ◽

...

Keyword(s):

Quality Control ◽

Single Cell ◽

Medical Personnel ◽

Cellular Heterogeneity ◽

Biological Information ◽

Cell Clusters ◽

The Neural Network ◽

Rnaseq Data ◽

Functional Features ◽

Cell Subpopulations

AbstractSingle-cell RNA sequencing (scRNAseq) is an essential tool to investigate cellular heterogeneity. Thus, it would be of great interest being able to disclose biological information belonging to cell subpopulations, which can be defined by clustering analysis of scRNAseq data. In this manuscript, we report a tool that we developed for the functional mining of single cell clusters based on Sparsely-Connected Autoencoder (SCA). This tool allows uncovering hidden features associated with scRNAseq data. We implemented two new metrics, QCC (Quality Control of Cluster) and QCM (Quality Control of Model), which allow quantifying the ability of SCA to reconstruct valuable cell clusters and to evaluate the quality of the neural network achievements, respectively. Our data indicate that SCA encoded space, derived by different experimentally validated data (TF targets, miRNA targets, Kinase targets, and cancer-related immune signatures), can be used to grasp single cell cluster-specific functional features. In our implementation, SCA efficacy comes from its ability to reconstruct only specific clusters, thus indicating only those clusters where the SCA encoding space is a key element for cells aggregation. SCA analysis is implemented as module in rCASC framework and it is supported by a GUI to simplify it usage for biologists and medical personnel.

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Spatially defined single-cell transcriptional profiling characterizes diverse chondrocyte subtypes and nucleus pulposus progenitors in human intervertebral discs

Bone Research ◽

10.1038/s41413-021-00163-z ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Yibo Gan ◽

Jian He ◽

Jun Zhu ◽

Zhengyang Xu ◽

Zhong Wang ◽

...

Keyword(s):

Single Cell ◽

Nucleus Pulposus ◽

Molecular Mechanisms ◽

Transcriptional Profiling ◽

Intervertebral Discs ◽

Cellular Heterogeneity ◽

Effector Functions ◽

Cartilage Endplate ◽

Intervertebral Disks ◽

Ivd Degeneration

AbstractA comprehensive understanding of the cellular heterogeneity and molecular mechanisms underlying the development, homeostasis, and disease of human intervertebral disks (IVDs) remains challenging. Here, the transcriptomic landscape of 108 108 IVD cells was mapped using single-cell RNA sequencing of three main compartments from young and adult healthy IVDs, including the nucleus pulposus (NP), annulus fibrosus, and cartilage endplate (CEP). The chondrocyte subclusters were classified based on their potential regulatory, homeostatic, and effector functions in extracellular matrix (ECM) homeostasis. Notably, in the NP, a PROCR+ resident progenitor population showed enriched colony-forming unit-fibroblast (CFU-F) activity and trilineage differentiation capacity. Finally, intercellular crosstalk based on signaling network analysis uncovered that the PDGF and TGF-β cascades are important cues in the NP microenvironment. In conclusion, a single-cell transcriptomic atlas that resolves spatially regulated cellular heterogeneity together with the critical signaling that underlies homeostasis will help to establish new therapeutic strategies for IVD degeneration in the clinic.

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