2. Fibroblast Cultures
Fibroblast cell cultures are the most basic and best established of all known culture types; therefore, they can be considered a model culture. These cells are found in almost all of the tissues of the body, including skin, bones, muscles and organs. Fibroblasts play a crucial role in maintaining the structural integrity of tissues and organs.
[20] Fibroblasts produce and secrete the extracellular matrix (ECM), a complex mixture of collagen, elastin and other proteins and carbohydrates, that forms the structural framework of tissues, providing them with necessary strength and flexibility. Fibroblasts also play a key role in early stages of wound healing and immune response by inducing receptor-linked chemokine synthesis
[20]. As a fundamental type of connective tissue cell, fibroblasts are more or less affected in most of the ACTDs; however, in the course of SSc
[11], DM
[8] and PM
[8] they play major roles. Briefly, in SSc, fibroblasts are targeted by immune cells which secrete profibrotic cytokines. These cytokines disrupt the balance of ECM production and degradation, causing fibroblasts to become dysfunctional and overproduce collagen and other proteins that form scar tissue. This excess collagen causes thickening and hardening of the skin, as well as damaging the blood vessels, muscles and internal organs
[21]. In DM and PM, the muscle tissue is invaded by T and B lymphocytes, similarly to SSc. But, instead of secreting profibrotic cytokines, they produce cytokines and antibodies that destroy myocytes and damage the tissue leading to muscle weakness and atrophy. Even though fibroblasts are not directly targeted in myositis, they may be damaged by infiltrating immune cells, releasing MDA-5 antigen and mobilizing anti-MDA-5 antibodies, causing an increase in inflammation and a characteristic skin rash
[22].
Establishing a primary cell culture of fibroblasts involves isolating these cells from tissue of interest and culturing them in a suitable growth medium. These tissues can be easily obtained through a biopsy or discarded surgical material. After acquisition, the collected material must be dissociated into smaller pieces, whether by using a sterile scalpel and/or by enzymatic digestion to release individual cells
[20]. Isolated cells should be rinsed with PBS, resuspended in DMEM with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic addition, and maintained in 5% CO
2 environment at 37 °C
[23,24,25][23][24][25].
One use of fibroblast cultures in the aforementioned diseases is to study the underlying mechanisms of the pathological processes involved in their development and progression
[4,26,27][4][26][27]. Chadli et al. conducted an experiment in which they used skin biopsies collected from patients diagnosed with early SSc to generate fibroblast cell culture. Biopsies were minced into small fragments, treated with dispase II to separate dermis from epidermis and then the dermis was treated with mixture of dispase II and collagenase II to free fibroblasts, which were cultured in standard conditions in DMEM
[26]. Subsequently, RNA was extracted from the cultured cells and micro-array was performed to measure the expression levels. The researchers found that fibroblasts derived from SSc skin biopsies retained most of the diseases’ molecular phenotype, therefore highlighting the value of phenotyped fibroblast cultures as a viable platform for SSc drug research
[26]. Another use of the fibroblast cell cultures in ACTD is in the development and testing of new therapies, such as Wu and colleagues’ research in which they used skin biopsies from patients with SSc in order to limit SSc pathogenesis through fibroblast store-operated Ca
2+ entry (SOCE) induced dedifferentiation
[28].
3. Synovial Fibroblast Cultures
Synovial fibroblasts (SF) are a type of cell found only in the stroma of the joint synovium and are its main component. Based on their localization in the synovium and their expression patterns, the cells can be distinguished into two groups, intimal and subintimal
[30][29]. The term “synovial fibroblasts” is a broad description that refers to both subpopulations; therefore, the intimal cells are also called type B synoviocytes or fibroblast-like-synoviocytes (FLS)
[31][30].
FBo
r the purposes of this work, both th groups of cells, despite distinct differences between them, will be called synovial fibroblasts, due to the fact that both subtypes are involved in the course of RA. Apart from being the basis of the stroma’s structure, synoviocytes’ main functions are producing components of the synovial fluid and immunosurveillance
[32][31]. Production of synovial fluid is crucial for the integrity of the cartilaginous tissue present in the joint, as well as its lubrication and mobility
[33][32]. SF play a key role in the development and progression of RA
[34][33] and osteoarthritis (OA)
[35][34], but, similarly to the chondrocytes which are found in the vicinity of SF, they are also indirectly affected in the course of other ACTDs that affect the joints with their symptoms, such as JIA, spondyloarthropathies and MCTD
[5,10,36][5][10][35]. At this point, it is important to note that OA is not considered an ACTD as it does not have an autoimmune or inflammatory background. However, OA is often investigated in ACTD research, whether as a control group in inflammatory joint damage research, in studies on joint tissue renewal therapies or as a starting point for research into the development of joint models essential for ACTDs with joint pathogenesis. In RA, due to chronic inflammation, synoviocytes undergo a series of morphological and physiological changes that result in acquiring a form of a constantly activated tumor-like RA-FLS. These cells, resistant to receptor mediated apoptosis and capable of invading other cells, are causing further inflammation and degradation of joints by the production of inflammatory factors, stimulation and the accumulation of immune cells
[34][33]. In OA, SF, mostly FLS, begin to intensively proliferate and differentiate into myofibroblast-like cells which synthesize immense amounts of rich in fiber ECM. This process leads to accumulation of ECM and joints fibrosis causing stiffness and chronic pain
[37][36].
The main sources of SF are aspirates of synovial fluid from joints
[38][37], fragments of tissue collected during joint surgeries
[39][38] and tissue harvested during arthroscopic synovectomy
[40][39]. SF culture from synovial fluid is conducted under the standard conditions using DMEM
[38][37]. 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][37] or filtering through a mesh
[39][38] and cultured in DMEM with 10% FBS and 1% antibiotic–antimycotic addition
[38,39,40][37][38][39]. 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. Wei et al. performed an experiment in which they used SF collected from RA patients and revealed that the upregulation of the neurogenic locus notch homolog protein 3 (NOTCH3) signaling pathway in SF plays a significant role in inflammation and tissue degradation processes during the course of RA
[41][40]. Moreover, SF cultures can aid in identifying novel therapy targets and testing new potential therapeutics. Three different research groups conducted experiments in which they used SF cultures derived from cells collected from RA patients has shown significant upregulation of fucosyltransferase 1
[42][41] and Yes-associated protein (YAP)
[43][42] expression, as well as a shift in glucose metabolism towards glycolysis
[44][43] suggesting them as novel targets in RA treatment strategies.
4. Chondrocyte Cultures
Chondrocytes are the only type of cell present in human cartilaginous tissue. They can be considered as a highly differentiated form of fibroblasts therefore their main function is synthesis of an ECM rich in proteoglycan and type II collagen
[45][44]. Moreover, chondrocytes maintain homeostasis in cartilage by alternating between anabolic and catabolic processes of creation and degradation of ECM proteins
[45][44]. The matrix’s functions vary based on its location within tissue or organ and the number of structural proteins it consists of. Nevertheless, its fundamental roles are supporting the growth of cells and tissues, modulation of intercellular interaction and separation of tissues. Additionally, ECM is able to alter cell behavior and growth by secretion of various factors which crucial in cellular growth, fibrosis and thereby wound healing. It has been proven that ECM plays an important role in cell differentiation, migration and even in gene expression
[46,47,48][45][46][47].
Chondrocytes are primarily affected in OA, which causes both cells and ECM to degrade, leading to the breakdown of joint cartilage and bone structure underneath
[49][48]. Along with the progression of the disease occurs chronic inflammation and tightening of space in the afflicted joint, due to the lack of cartilaginous tissue and growth of pathological bone structure, causing stiffness, swelling and pain
[50][49]. Chondrocytes are also indirectly affected in the course of other ACTD such as RA, JIA, MCTD and systemic lupus erythematosus (SLE) due to the persistent, prolonged inflammation that causes joint stiffness, swelling and pain, which may lead to ECM disbalance and degradation of chondrocytes
[51,52,53,54][50][51][52][53].
Chondrocyte cell cultures are widely used in OA research, both in its etiology and treatment. In the case of ACTD studies, OA is used as a control model due to the lack of an autoimmune/inflammatory background
[55][54]. They can be also used in treatment research by testing cellular response to drugs
[56,57,58][55][56][57] inhibiting the diseases progress using genetic engineering
[59,60,61][58][59][60] or by coculturing chondrocytes with other cells to enhance therapy effectiveness in articular chondrocyte implantation
[62][61].
The main source of human cartilage cells is total knee arthroplasties (TKAs Partial knee arthroplasty is an equally valid source of cells acquisition
[63][62]. Chondrocytes can be isolated from the afore-mentioned harvested tissue and be used in setting up a primary chondrocyte cell culture in standard conditions using DMEM medium. In order to do so, cartilage must be minced or cut into very small pieces and treated with type II 0.02% collagenase at 37 °C for 4 h
[64,65][63][64]. The concentration of collagenase may be lower, but the digestion time should be adequately prolonged
[45,66][44][65]. However, while being cultured in 2D in vitro conditions, chondrocytes undergo a process of dedifferentiation altering their appearance into one resembling a fibroblast. Recently it has been claimed that this change also affects the expression levels of various genes and proteins, making chondrocyte cultures a less reliable tool for research
[67][66]. Chondrocyte cultures can be used to study the etiology of RA, as described in an experiment by Andreas et al., in which they created a model consisting of postmortem-acquired human chondrocytes, supernatants from cultures of RA SF and alginate. By stimulating chondrocytes with RA SF culture supernatants, they mimicked the RA joint environment, which allowed them to identify the key regulatory molecules driving cartilage destruction
[68][67]. Chondrocyte cultures also allow for the study of changes in cell physiology during the course of the disease. By constructing coculture models consisting of either three
[69][68] or two
[70][69] types of cells deriving from patients or animals, respectively, researchers were able to recreate the joint environment, which allowed the first group to establish a complete model for cartilage destruction
[69][68] and the second group to create a perfusion culture system
[70][69]. Chondrocyte cell cultures can also be used to observe the effects of illness on gene expression levels and gene involvement in RA pathogenesis, as described by Barksby et al. In their experiment, the researchers cultured two types of cells, chondrocytes acquired from patients and commercially acquired human chondrosarcoma cells; both were cultured in standard conditions with use of serum-free DMEM. These cells were stimulated with a combination of interleukin 1 (IL-1) and oncostatin M (OSM) to mimic cytokine environment of RA synovial fluid. Subsequently, researchers isolated RNA from the cells and performed micro-array analysis, as well as a real-time PCR, which showed an overexpression of multiple genes, such as matrix metalloproteinase 1 (MMP1), a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and pentraxin-related protein 3 (PTX3)
[71][70].