Cell Cultures in the Autoimmune Connective Tissue Diseases

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1		Adam Ejma-Multański	--	3473	2023-10-31 09:04:22	\|
2	update references and layout	Rita Xu	-6 word(s)	3467	2023-10-31 09:08:26	\| \|
3	update description	Rita Xu	+ 4 word(s)	3471	2023-11-24 07:29:08	\|

This entry is adapted from the peer-reviewed paper 10.3390/cells12202489

Cell cultures are an important part of the research and treatment of autoimmune connective tissue diseases. By culturing the various cell types involved in autoimmune connective tissue diseases (ACTDs), researchers are able to broaden the knowledge about these diseases that, in the near future, may lead to finding cures. Fibroblast cultures and chondrocyte cultures allow scientists to study the behavior, physiology and intracellular interactions of these cells.

ACTD cell cultures systemic sclerosis lupus erythematosus

1. Introduction

Autoimmune connective tissue diseases (ACTDs) are a group of complicated multisystem disorders in which patient’s immune system cells attack tissues such as joints, tendons, skeletal muscles, neural connections, cartilage, bones and other connective tissues ^[1]. Even though these diseases differ from each other in their symptoms as well as in affected cells, they all share the same pathological mechanism, which manifests as a prolonged state of inflammation in the affected area or areas, leading to the degeneration and degradation of afflicted structures ^[2]. The etiology of ACTDs remains mostly undiscovered with suspected involvement of genetic, hormonal and environmental factors ^[3]. Because of the unknown etiology and ACTDs’ multisystemic nature, proper diagnosis and treatment is a major challenge for healthcare systems. Therefore, cell cultures are one of the tools used to understand the molecular basis/mechanisms of/in ACTDs. Due to the lack of uniformed diagnosis criteria and the amount of ACTDs, it would be impossible to describe the cell culture types used in the research on all of them in one paper. So the authors have focused on few selected ACTDs, and the culture types used in research on them. These diseases are presented in Table 1.

Table 1. List of described ACTDs, tissues they affect, type of cell cultures that might be used in research on particular diseases, immune elements involved in pathological processes and their immunopathogenesis.

ACTD	Affected Tissue	Type of Cell Culture	Involved Immune Elements	Immunopathogenesis
Dermatomyositis (DM)	Muscles, skin ^[4].	2D and 3D fibroblast cultures and skin models.	Myositis-specific antibodies: nuclear matrix protein 2 (anti-NXP-2), transcriptional intermediary factor 1 γ (anti-TIF1-γ), melanoma differentiation-associated protein 5 (anti-MDA-5), small ubiquitin-like modifier activating enzyme 1 (anti-SAE-1), subunit of nucleosome remodeling deacetylase complex (anti-Mi-2) ^[4].	Characterized by a chronic inflammatory process affecting the skin and muscles. Infiltration of the affected tissues by immune elements leads to tissue damage and inflammation. Also associated with microangiopathy in affected tissues, which contributes to skin changes, muscle weakness and other clinical features ^[4].
Juvenile idiopathic arthritis (JIA)	Joints, cartilage, bones ^[5].	2D and 3D fibroblast and chondrocyte cultures.	Macrophages and T lymphocytes ^[5].	Overactive immune response, characterized by an influx of T cells and macrophages into the synovium and the release of proinflammatory cytokines, which promotes inflammation and tissue damage in the joints. Various subtypes with significant differences in pathogenesis ^[5].
Lupus erythematosus (LE)	Various, including joints, blood cells, brain, heart, skin, kidneys, lungs ^[6].	2D and 3D fibroblast, chondrocyte and cardiomyocyte cultures, peripheral blood mononuclear cells (PBMC) cultures, skin models.	Defective autoantibodies recruiting T and B lymphocytes ^[6].	Dysregulated immune response, leading to the activation of various components of the immune system. Imbalance between proinflammatory and regulatory immune responses. Production of autoantibodies which target and attack the body’s own tissues. Formation of immune complexes by antibodies with their target antigens, complexes can deposit in various tissues leading to inflammation and tissue damage ^[6].
Mixed connective tissue disease (MCTD)	Various, including skin, muscles, small capillary vessels, lungs, kidneys ^[7].	2D and 3D fibroblast, chondrocyte and endothelial cell cultures, PBMC cultures.	Anti-U1-RNP antibodies ^[7].	Immunological imbalance features similar and overlapping with lupus, systemic sclerosis and polymyositis ^[7].
Polymyositis (PM)	Muscles ^[8].	2D and 3D fibroblast and myocyte cultures.	CD8+ T cells ^[8].	Infiltration of CD8+ T cells into the muscle tissue, which recognize and attack muscle fibers, leading to muscle inflammation and damage. Persistent inflammation interferes with the muscle regeneration processes. As muscle fibers are damaged and replaced by fibrous tissue, muscle strength and function are compromised ^[8].
Rheumatoid arthritis (RA)	Joints, cartilage and bones ^[9].	2D and 3D fibroblast and chondrocyte cultures.	CD4+ T helper cells, rheumatoid factor (RF), anti–citrullinated protein antibodies (ACPA) ^[9].	Dysregulation of immune response with activation of CD4+ T helper cells and production of proinflammatory cytokines. Synthesis of RF and ACPA autoantibodies, leading to inflammation and tissue damage, mostly in joint areas. Formation and deposition of immune complexes in tissue which contribute to inflammation. Cytokine-stimulated bone erosion and deformation caused by osteoclasts ^[9].
Spondyloarthropathies	Joints of the vertebral column ^[10].	2D and 3D fibroblast and chondrocyte cultures.	Human leukocyte antigen B27 (HLA-B27) antibodies ^[10].	Inappropriate immune response, characterized by the activation of immune cells, leading to chronic inflammation and infiltration of the affected joints, including the spine and peripheral joints. Overproduction of proinflammatory cytokines contributing to enthesitis and tissue damage. Various subtypes with significant differences in pathogenesis ^[10].
Systemic sclerosis (SSc)	Skin, soft tissue organs, blood vessels, joints ^[11].	2D and 3D fibroblast, PBMC, mesenchymal stem cell (MSC) and endothelial cell cultures, skin models.	T cells, CD4+ T cells ^[11].	Persistent activation of fibroblasts, leading to imbalance in extra cellular matrix (ECM) production and degradation processes resulting in fibrosis. Activation of endothelial cells leads to an abnormal release of vasoactive factors, resulting in blood flow changes and vessel dysfunction. Infiltration of the affected tissues by inflammatory cells and release of proinflammatory cytokines and chemokines result in tissue damage ^[11].
Vasculitis	Various blood vessels ^[12].	2D and 3D endothelial cell cultures, PBMC cultures.	Anti-neutrophil cytoplasmic antibodies (ANCAs) ^[12].	Dysregulation of the immune system, leading to the activation of immune cells and production of proinflammatory cytokines and chemokines resulting in damage to vessels. Activation of endothelial cells, resulting in the promotion of immune cell infiltration. Various subtypes with significant differences in pathogenesis ^[12].

Cell culture development began in the early 1900s; however, their true potential was discovered and advanced in late 1940s when they were used to grow polioviruses in the production of the polio vaccine ^[13]. Conducting an experiment with cell cultures allows scientists to study the in vitro morphology, physiology and genetics of a single or few types of cells of interest. While cell cultures are a powerful tool in research, it is important to remember their limitations and requirements. First of all, working with tissue cultures requires strict conditions, such as CO₂ levels, temperature, etc. Secondly, in order to grow cells must be placed in dedicated media which maintain their physiological state and functionality ^[14]. The media which are used in the cell cultures are listed in Table 2. Depending on the type of cell culture, its development might be a long and laborious process, which is why researchers often buy commercially prepared cell lines to hasten the experiment.

Table 2. List of medias used in the described cell cultures with their composition and application.

Medium Name	Composition	Application
Dulbecco’s Modified Eagle Medium (DMEM)	Glucose, amino acids, vitamins, minerals and a buffering agent to maintain a constant pH. Available in numerous modifications, including increased glucose concentration, sodium pyruvate addition or prolonged shelf life ^[15].	A wide range of cell types, including fibroblasts, epithelial cells, smooth muscle cells, neurons, glial cells and certain cell lines ^[15].
Ham’s F-12	Modification of Eagle Medium similar in composition to DMEM but with a higher concentration of bicarbonate, which helps to regulate the pH of the medium and maintain proper osmotic pressure ^[15].	A wide range of cell types, including epithelial cells, fibroblasts, neurons, endothelial cells, hepatocytes, adipocytes and cells that are sensitive to changes in osmotic pressure ^[15].
M199	Modification of Eagle Medium similar in composition to DMEM but with high concentration of inorganic salts, which help to maintain the proper osmotic pressure and provide a stable environment for the cells ^[16].	Fibroblasts, endothelial cells, epithelial cells, hybridoma cells, hepatocytes, smooth muscle cells and cells sensitive to osmotic pressure changes ^[16].
α-Modified Eagle Medium (α-MEM)	A modification of the original minimum essential medium (MEM) with high concentration of vitamins and low concentration of pyruvate ^[16].	Fibroblasts, epithelial cells, stem cells, hybridoma cells, adipocytes and osteoblasts ^[16].
Roswell Park Memorial Institute (RPMI) 1640	A modification of MEM supplemented with additional compounds, including HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), which helps to maintain the pH of the medium in the presence of carbon dioxide and sodium bicarbonate, which helps to buffer the medium ^[15].	Lymphocytes, cancer cells, hybridoma cells, adipocytes, fibroblasts and epithelial cells ^[15].
Human Endothelial Serum Free Medium (Human Endothelial SFM)	Serum-free medium with a range of supplements, including growth factors, amino acids, vitamins and trace elements that are essential for EC growth, such as Vascular Endothelial Growth Factor (VEGF), Basic Fibroblast Growth Factor (bFGF) and Epidermal Growth Factor (EGF) ^[17].	Human ECs, such as human umbilical vein endothelial cells (HUVECs), human dermal microvascular endothelial cells (HDMECs), human pulmonary microvascular endothelial cells (HPMECs) and human coronary artery endothelial cells (HCAECs) ^[17].
Endothelial Cell Growth Medium (EGM)	Variety of nutrients, growth factors and supplements that are necessary for the growth and survival of ECs, which can include growth factors such as VEGF, bFGF and EGF, as well as other components, such as hydrocortisone, ascorbic acid and heparin ^[18].	It is important to note that EGM medium is formulated for specific types of ECs. Therefore, different ECs will require different EGMs. Some of the cells that can be cultured in this medium are HUVECs, human microvascular endothelial cells (HMVECs), human pulmonary artery endothelial cells (HPAECs), HDMECs, HCAECs and human retinal microvascular endothelial cells (HRMECs) ^[18].

Connective tissue is a type of tissue that is present in every physiological system in the human body. It acts as a scaffolding for other tissues and organs, supports their functions, protects them against harmful factors and helps to repair damaged tissues ^[19]. During the course of ACTD-afflicted connective tissue with ongoing inflammation and, very often, fibrosis, is unable to perform its functions, thus starting a domino effect leading to deterioration of other tissues and organs.

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% CO₂ environment at 37 °C ^[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]. 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 Ca²⁺ 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 ^[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) ^[30]. Both 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 ^[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 ^[32]. SF play a key role in the development and progression of RA ^[33] and osteoarthritis (OA) ^[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]^[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 ^[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 ^[36].

The main sources of SF are aspirates of synovial fluid from joints ^[37], fragments of tissue collected during joint surgeries ^[38] and tissue harvested during arthroscopic synovectomy ^[39]. SF culture from synovial fluid is conducted under the standard conditions using DMEM ^[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 ^[37] or filtering through a mesh ^[38] and cultured in DMEM with 10% FBS and 1% antibiotic–antimycotic addition ^[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 ^[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 ^[41] and Yes-associated protein (YAP) ^[42] expression, as well as a shift in glucose metabolism towards glycolysis ^[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 ^[44]. Moreover, chondrocytes maintain homeostasis in cartilage by alternating between anabolic and catabolic processes of creation and degradation of ECM proteins ^[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 ^[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 ^[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 ^[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 ^[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 ^[54]. They can be also used in treatment research by testing cellular response to drugs ^[55]^[56]^[57] inhibiting the diseases progress using genetic engineering ^[58]^[59]^[60] or by coculturing chondrocytes with other cells to enhance therapy effectiveness in articular chondrocyte implantation ^[61].

The main source of human cartilage cells is total knee arthroplasties (TKAs Partial knee arthroplasty is an equally valid source of cells acquisition ^[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 ^[63]^[64]. The concentration of collagenase may be lower, but the digestion time should be adequately prolonged ^[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 ^[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 ^[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 ^[68] or two ^[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 ^[68] and the second group to create a perfusion culture system ^[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) ^[70].

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