Human fibroblast-like synoviocyte isolation matter: a comparison between cell isolation from synovial tissue and synovial fluid from patients with rheumatoid arthritis

Abstract

OBJECTIVE:Cell culture technology has become a popular method in the field of cell biology, pharmacology, and medical researches.Primary cells represent the normal physiological condition of human cells. Fibroblasts are the most common native cells of connective tissue that play a crucial role in the entire pathogenesis of various disorders, such as rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLSs), which overlie the loose connective tissue of the synovial sublining, are known to be the central mediators of joint damage. The most routine approach for the isolation of FLS is an enzymatic digestion of synovial tissue. This experimental study is designed to introduce an easy, fast, and high-throughput method compared with enzymatic digestion for isolation of FLS. METHODS:The synovial tissue and synovial fluid (SF) samples were collected from eight patients with RA who underwent routine knee replacement surgery. Synovial tissue was incubated with collagenase VIII enzyme, while SF was washed with a similar volume of phosphatebuffered saline. The cells were further subcultured and stored based on the standard protocols. The purity of isolated synoviocytes was confirmed using flow cytometry analysis.RESULTS: Isolation of FLS from SF was more successful with a faster rate, 3-5 days after culture. The morphological assessment and flow cytometry analysis confirmed the purity of SF-derived cells in passage 4.CONCLUSIONS: SF could be a more accessible source of FLS than synovial tissue. Obtaining primary FLS from SF is a simple, fast, and cost-effective way to have a large-scale cell during a short time.

Inflammation‐Targeting Mesenchymal Stem Cells Combined with Photothermal Treatment Attenuate Severe Joint Inflammation

“…Human FLSs were isolated from the synovial tissues of patients with RA (age range: 32–59 years), using enzymatic dispersion, as previously described. [ 55 ] Informed consent was obtained from all patients, and this study was approved by the Human Subject Study Ethics Committee of Eulji University. The FLSs were maintained in Dulbecco's modified Eagle's medium (DMEM; 11965‐092, Gibco) with 10% fetal bovine serum (FBS; 26140, Gibco) and 1% antibiotics (10378016, Gibco).…”

Assessment of disease activity in patients with rheumatoid arthritis using plasma tumour M2-pyruvate kinase test

“…Cell pellet was then resuspended for the next passage expansion. The media used for cell culture was changed every 3-4 days ( 27 ), and cells in the passage of 3-5 were used for all experiments. Cells used for the experiments were confirmed as FLS by flow cytometry, which was determined as positive for fibroblast marker of CD44 and CD90 and negative for macrophage marker of CD14 (all from BD Biosciences, San Diego, CA, USA) ( 28 – 30 ).…”

Cell Cultures as a Versatile Tool in the Research and Treatment of Autoimmune Connective Tissue Diseases

“…The main sources of SF are aspirates of synovial fluid from joints [38], fragments of tissue collected during joint surgeries [39] and tissue harvested during arthroscopic synovectomy [40]. SF culture from synovial fluid is conducted under the standard conditions using DMEM [38].…”

“…The main sources of SF are aspirates of synovial fluid from joints [38], fragments of tissue collected during joint surgeries [39] and tissue harvested during arthroscopic synovectomy [40]. SF culture from synovial fluid is conducted under the standard conditions using DMEM [38]. Deriving a cell culture from tissue fragments, either harvested during surgeries or synovectomy, first requires a manual fragmentation and digestion with collagenase in DMEM for 2 h at 37 • C. When freed from the fibrous scaffold, tissues can be harvested by centrifuging [38] or filtering through a mesh [39] and cultured in DMEM with 10% FBS and 1% antibiotic-antimycotic addition [38][39][40].…”

“…SF culture from synovial fluid is conducted under the standard conditions using DMEM [38]. Deriving a cell culture from tissue fragments, either harvested during surgeries or synovectomy, first requires a manual fragmentation and digestion with collagenase in DMEM for 2 h at 37 • C. When freed from the fibrous scaffold, tissues can be harvested by centrifuging [38] or filtering through a mesh [39] and cultured in DMEM with 10% FBS and 1% antibiotic-antimycotic addition [38][39][40]. Synovial fibroblasts play an important role in the pathogenesis of various ACTDs, such as RA; therefore, their cultures are a valuable tool for studying the pathogenesis of these diseases.…”