Development of scRNA-seq technique contributes a big step for cellular biology. Preparation of qualified single-cell suspension is a precondition for the platform. It’s full of challenge to offer eligible samples from dense and tense musculoskeletal tissues, like bone, cartilage, nucleus pulposus, tendons, etc. In the present study, we aimed to provide standard and specific protocols for preparing qualified single-cell suspensions from human degenerated nucleus pulposus, ossifying posterior longitudinal ligaments and knee articular cartilage with OA. As a result, the protocols for the former two types of tissues could be applied to output qualified samples for following scRNA-sEq. However, dissociated cell suspensions from knee articular cartilage were unqualified due to either excessive aggregates and doubles or severely damaged cell viability, which needed more efforts in the future.
Nucleus pulposus, the inner part of intervertebral disc, performs critical biomechanical functions [8–10]. Accumulated evidences supported that the nucleus pulposus degenerated at early stage of degenerative disc disease [11–13], which is a common cause of disability and results in a huge socioeconomic impact world widely [14]. The etiology as well as pathology is complex and multifactorial [9, 15]. Although the underlying pathology has been approached from a variety of ways [9], a systematically study to get a panorama is still missing. The single-cell platform provided a revolutionary way to solve this problem, but there was no related report till now. Therefore, to harvest qualified single-cell suspension would be the first step. In previous studies, NP was usually dissociated via various enzymatic methods and the treatment duration was from 30 min to overnight, which was summarized detailly in Juliana’s paper [16]. As is well known, long time and intense enzymatic treatment would increase cell yield while damage cell viability, vice versa. And the lack of systematic quality control made it hard to be directly used in single-cell isolation.
Pronase, a mixture of several nonspecific endo- and exoproteases, has been wildly used in isolating cells from NP [17]. It was reported that Pronase treatment might shorten the required digestion time [16]. Based on these researches, we combined Pronase with the S-D solution for single-cell isolation from NP in this study and obtained highly qualified single-cell suspensions possessing abundant cell yield, high cell viability, and few cell aggregates or doublets. In conclusion, the enzymatic treatment protocol for human NP was validated for single-cell platform.
The posterior longitudinal ligament runs entire length of the spine. Ossification of the posterior longitudinal ligament (OPLL), defined as an ectopic ossification (calcification), would lead to paralysis of extremities and disturbances of motility [18]. Up to now, the osteo-lineage derivation, cell atlas transformation and molecular regulation have never been elucidated, which retarded the development of etiological treatment [18–20]. Single-cell platform, taking advantage of horizontal cell atlas and vertical trajectory analysis, would broaden the horizons in this field. Normal posterior longitudinal ligament is fibrous connective tissue. However, the ossifying posterior longitudinal ligament is a mixture of ligament, bone and probably cartilage as a result of endochondral ossification process [21, 22]. It’s full of challenge to ascertain one digestion medium to dissociate mixed tissues. Collagenase type Ⅰ, frequently used in digestion of bone and tendons, was combined with the S-D solution in the present study. Moderate cell yield, high cell viability and low content of cell aggregates or doublets were realized using the present protocol. Although scRNA-seq was conducted, the cell heterogeneity should be inspected carefully by data analysis. In particular, the quantity and variety of bone derived cells should be noted in consideration of lots of bone-like tissue remnants. More experiments will be carried out to raise the cell yield but not compromise on cell viability.
Articular cartilage is specialized connective tissue of diarthrodial joints and functions as a load-bearing, low-friction, and wear-resistant surface to facilitate joint movement [23]. As it is lack of self-regenerative ability, articular cartilage is one of the most common degenerative tissues. Osteoarthritis (OA) is the primary joint disorder and a major cause of disability, affecting 303 million people globally in 2017 [24, 25]. The etiology, pathology and regenerative therapy have been wildly studied [26–29]. Also utilizing the single-cell platform, there were two nice reports that systematically uncovered the cell atlas and transcriptional regulation mechanisms of OA [30, 31]. As for other related studies, several reports showed the single-cell profiling results of developing limbs, including growth plate cartilage as well as articular cartilage [32–35]. The single-cell isolation methods used in the above studies were summarized in Table 1. In Quanbo Ji’s research, the articular cartilage was obtained from 10 patients but only 1600 cells were sequenced, suggesting a too moderate digestive efficiency. In Fiorella Carla Grandi’s study, the primary chondrocytes were delivered to culture and treatment in vitro before scRNA-seq, the quality control of isolated primary single cells was not reported. And in other researches, samples were obtained from mice but not human.
Table 1
Summary of Single-cell Isolation Protocols for Cartilage in Reported Studies Using scRNA-seq Technique
Reference | Tissue | Species | Treatment | Number of profiling cells |
Quanbo Ji, et al. [30] | Cartilage from 10 knee OA patients | Human | 0.25% Trypsin-EDTA for 30 min and then 0.2% type II collagenase (Sigma-Aldrich) for 4 h. | 1600 |
Fiorella Carla Grandi, et al. [31] | OA samples from patients undergoing total joint replacement | Human | Collagenase II and IV (2.5 mg/ml each; Worthington Biochem) overnight. | Not reported |
Junxiang Li, et al. [32] | The distal cartilage structure at postnatal day 7. | Mouse | 0.2% collagenase II for 2 hours | 217 |
Vikram Sunkara, et al. [33] | Femur of 12 weeks old mice | Mouse | 10 mg/ml Collagenase II (Nordmark) supplemented for 4 h | 7133 |
Koji Mizuhashi, et al. [34] | Femur growth plate cells at postnatal day 2. | Mouse | Liberase TM (Sigma/Roche) for unclear time | 18,000 |
Natalie H Kelly, et al. [35] | Hindlimb bud of embryonic mice | Mouse | Collagenase Type II (Worthington-biochem, Lakewood, NJ) and pronase (EMD Millipore, Billerica, MA) in 15 min increments with agitation for up to 1 h | 9466 |
Collagenase type II was wildly used in dissociation articular cartilage, which was added with the S-D solution in this study. Using the rapid protocol, excessive live cell aggregates suggested insufficient enzymatic efficiency but longer duration of treatment significantly damaged cell viability with plenty of dead cell aggregates. Different from insufficient digestion, the dead cells could be aggregated into various sizes by released DNA. DNase could be used to degrade DNA and reduce dead cell aggregation, and FACS or MACS could be used to remove the dead cells. However, the potential loss of cell heterogeneity might mislead the data analysis. So longer enzymatic duration was not a better choice.
For hard dissociated tissues, single-nucleus RNA-sequencing (snRNA-seq) is an appealing alternative choice. In this method, a mild and quick nuclear dissociation protocol is used to isolate and sequence RNA within the nucleus, which minimizes technical issues that can arise from common dissociation protocols [36–37]. More detailed explorations will be performed in the future.