In order to ensure experimental reproducibility, BMDMs closely resemble native in vivo macrophage characteristics and functions, thereby avoiding culture-induced variations inherent to cell lines.

BMDMs were isolated from mouse femurs and tibias using a standard bone marrow harvest and erythrocyte lysis protocol (28, 29). BMDMs differentiate from bone marrow monocytes (BMMs); however, the isolation of bone marrow monocytes is technically challenging and requires fresh preparation for each experiment. Mature BMDMs can only be obtained after a 5–7 day induction period with M-CSF or equivalent factors (e.g., secretory factors from L929 cells) in the extracellular milieu, which significantly extends experimental timelines (30, 31).

Macrophages produced in human bone marrow are terminally differentiated, non-proliferative cells that resemble macrophages but are unable to passage. However, because of ethical limitations, a lack of therapeutic necessity, and logistical difficulties in procurement, their usage in research is still restricted.

PBMCs can be isolated from porcine, bovine, rabbit, or piscine sources using standardized density gradient centrifugation protocols. For bovine PBMCs isolation, representative methods involve heparinized blood collection, density-based fractionation with species-specific separation media, and adherence-based purification under controlled culture conditions (35).

It is challenging to directly isolate sufficient macrophages from human tissues. Since human blood monocytes are easily obtained in high quantities and have the capacity to develop into macrophages in vitro, MDMs offer a great substitute. Procedure for MDMs in humans: Use density gradient centrifugation to separate PBMCs. PBMCs should be resuspended in full macrophage culture media. Adherent MDMs remain after non-adherent cells are removed.

The pulmonary microenvironment contains at least three different types of macrophages: AMs, interstitial lung macrophages, and bronchial macrophages (36). The majority of the innate immune cells in distal lung tissues are alveolar macrophages AMs that reside on the alveolar luminal surface and have cytoplasm that is enriched with lysosomes and lamellar bodies. These cells constitutively express core surface markers such as CD206, CD169, and F4/80, and they are involved in the phagocytosis of pathogens and the efferocytosis of apoptotic cells (37, 38). AMs are a great model for researching bacterial infections since they are the first line of defense against lung pathogens. Their sources, which include humans and animals, make their isolation quite simple.

Compared to other macrophage subtypes, PMs possess significant immunoregulatory functions, improved functional/phenotypic stability, and superior cytokine expression (41). PMs are ideal for short-term phagocytosis functional tests because they exhibit high expression of surface markers such as F4/80, CD11b, and CD14 (42, 43).

KCs are tissue-resident macrophages (TRMs) that are exclusive to the liver and are identified by the surface expression of CD11b, F4/80, TIM4, and CLEC4F. These cells, which make up more than 80% of the body’s resident macrophage pool, are produced from blood monocytes that adhere to hepatic sinusoids. They support hepatic repair and regeneration and preserve inflammatory homeostasis (47). Through scavenger receptor-dependent pathways, KCs eliminate gut-derived bacterial products, senescent blood cells, apoptotic hepatocytes, and immunological complexes, reducing the severity of viral infections in the liver (48, 49). Through a variety of mechanisms, KCs are activated upon hepatic damage and are essential in a number of liver disorders. Research has shown that in mice fed a methionine/choline-deficient (MCD) diet, KCs are depleted during the early stages of non-alcoholic steatohepatitis (NASH), and that Ly6C monocyte-derived macrophages subsequently take their position as the predominant population (50). KCs have a strong capacity for self-renewal and passaging in vitro (51).

Primary KCs were isolated from rodent liver using established enzymatic digestion and density gradient centrifugation methods (51), with purification via selective adhesion. Cell identity was confirmed by immunophenotyping using macrophage-specific markers (CD68、CD163、CK18、CD31、α-SMA).

Under physiological conditions, the blood-brain barrier (BBB) restricts cellular and molecular transit into the brain, limiting macrophage infiltration. Microglia, however, serve as the resident macrophages of the central nervous system (CNS), ubiquitously distributed throughout the brain and spinal cord. They constitute 5-20% of all glial cells in the CNS, with their physiological density and function being essential for maintaining neural homeostasis (7). Beyond phagocytic capabilities, microglia play pivotal roles in CNS development and homeostasis through immune surveillance, neurogenesis support, synaptic refinement, axonal growth guidance, injury repair, and neurotrophic factor secretion (52, 53). Microglial polarization shares similarities with but exhibits greater specificity than the classical M1/M2 dichotomy: M1-polarized microglia predominantly exert pro-inflammatory and neurotoxic effects, while their M2-polarized counterparts demonstrate anti-inflammatory and neuroprotective functions. The M2 phenotype is further subclassified into three distinct subsets (M2a, M2b, and M2c), each possessing unique molecular signatures and biological functions (54, 55).

Primary microglia exhibit a complicated morphology with cytoplasmic vacuolization and short processes, and they proliferate slowly. Myeloid markers CD68, CD11b, and CD14 are first expressed by them, but with prolonged in vitro culture, marker expression gradually declines (56). Neonatal microglia were isolated from brain tissue through mechanical dissociation and orbital shaking separation of mixed glial cultures (57), followed by phenotypic validation with standard markers.

A special immune system found in the placenta strikes a balance between the mother’s protection against infections and the fetus’s immunological tolerance. Decidual macrophages and Hofbauer cells are two different groups of placental macrophages (58). As demonstrated by immunohistochemical research, the placenta’s macrophage proportions fluctuate dynamically throughout pregnancy, from around 50% to 20% prior to birth (59, 60). Typically, isolation procedures include density gradient centrifugation (Ficoll or Percoll), enzymatic digestion (collagenase, DNase, and/or trypsin), and positive/negative selection employing antibodies that target CD68, CD10, or CD14 or take advantage of adhesion qualities (61). However, the translational applicability of mouse models is restricted due to the phenotypic differences between human and rat placental macrophages (62), which in turn limits the functional characterization of human placental macrophages. In view of this, the majority of research on human placental macrophages focuses on tissue section immunohistochemical analysis, with functional studies being comparatively rare (61).

In teleost fish, the head kidney’s primary purpose in the early stages of life is excretion. As an adult, it develops into a vital hematological and immunological organ that functions similarly to human bone marrow and is the genesis of macrophages and phagocytic activity (63). HKMs have a distinct polarization profile from the mammalian M1/M2 paradigm. M1-polarized HKMs express CXCR3.1 and iNOS, but they do not have traditional M2 polarization; instead, they have a regulatory phenotype that is dominated by CXCR3.2 and Arg2 (64). Numerous fish species, including salmonids, Atlantic cod, and southern bluefin tuna, have well-established macrophage isolation and culture methods, which have made it possible to conduct in-depth study in fish immunology (65–68).

In order to maintain intestinal microenvironmental equilibrium, IMs are essential for differentiating between dangerous microorganisms and innocuous antigens, such as dietary proteins and resident commensal flora (69). Lamina propria macrophages (LPMs) and muscularis macrophages (MMs) are two types of IMs. LPMs perform essential innate immune effector functions and act as the main sentinels against pathogens that penetrate the epithelial barrier. Living close to the enteric nervous system, MMs modulate peristalsis, protect tissue during inflammation and stress, and maintain enteric neurons by providing trophic and neuroprotective support (70, 71). With the expression of specific receptors such as Toll-like receptors (TLR3-TLR9), CD36, NOD-like receptors, and TREM2, LpMs have strong phagocytic and bactericidal action against pathogens (70). The boosted expression of tissue-protective genes (including Retnla, Mrc1, and CD163) distinguishes MMs from their LPMs in terms of transcription and morphology (70). Weigmann et al. used traditional enzymatic digestion to create an isolated procedure for mouse intestine lamina propria mononuclear cells (72). In order to isolate rat intestinal LPMs, Ana et al. improved this method using mechanically aided enzymatic dissociation, leading to increased purity and viability with less tissue input (73).

Numerous techniques for producing iPSCs from somatic cells have surfaced since Yamanaka et al. converted mouse fibroblasts into iPSCs by adding the OSKM transcription factors (OCT4, SOX2, c-MYC, and KLF4) (98). Through reprogramming, human-iPSCs are created from terminally differentiated somatic cells while maintaining the ability to self-renew (99). iPSCs function and develop similarly to embryonic stem cells (ESCs); they can differentiate into any type of cell, including macrophages, and have an infinite capacity for proliferation (100). Furthermore, iPSCs synthesis does not require embryonic material, in contrast to ESCs. By making it possible to derive stable, uniform, and genetically defined macrophages from pre-engineered genotype-specific iPSCs, IPSDMs solve important issues in macrophage procurement (101). Investigating immune response mechanisms, activation/polarization dynamics, macrophage-specific functions, and pathologies linked to macrophages (such as inflammatory disorders and cancer immunotherapy) is made possible by this platform.

The RAW264.7 cell line was first established in 1976 by Raschke et al., isolated from a male BALB/c mouse injected with Abelson murine leukemia virus (MuLV) (18). These cells exhibit a round or oval morphology with dark pigmentation, strong adherence, pseudopod extension, enhanced migratory capacity, and robust phagocytic activity. Due to their capabilities in pinocytosis and phagocytosis, RAW264.7 cells are widely utilized as a macrophage model in inflammation, immunology, apoptosis, and cancer research (105). While this cell line serves as a practical tool for preliminary screening of macrophage-related factors and functions, prolonged passaging may lead to progressive depletion of macrophage-specific gene/protein expression and impaired immune functionality compared to primary macrophages or in vivo models. Research indicates that RAW264.7 macrophages exhibit increased expression of genes including HIF1α, ITGAL, and CD86 after 50 passages, whereas changes in ARG1, TRF2, and IRF8 expression start to occur as early as 15 passages (106).

In 1968, a BALB/c mouse’s reticulum cell sarcoma gave rise to the J774A.1 cell line (17). In addition to retaining many of the functional characteristics of normal macrophages, such as phagocytosis (e.g., engulfing bacteria, apoptotic cells, or fluorescent-labeled particles), activation (inducible to M1/M2 polarization in vitro), and secretory activity (e.g., production of IL-1β and high levels of lysozyme), J774A.1 also demonstrates relative stability and ease of proliferation in culture (17, 107). LPS, dextran sulfate, pure protein derivative (PPD), and other substances suppress its proliferation (108). It is noteworthy that during in vitro culture, this cell line does not require external addition of M-CSF or other growth hormones. It is unclear, nevertheless, if J774A.1 makes M-CSF on its own to get around this requirement or if its tumor-derived origin gives it intrinsic proliferative ability that is not dependent on M-CSF signaling (109).

After methylcholanthrene-induced lymphoma development, DBA/2 mice were employed to create the murine monocyte/macrophage-like cell line P388D1 (110). IL-1 and lysozyme production in response to lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) stimulation (111), phagocytosis of zymosan and latex microspheres (112), and surface immunoglobulin (sIg) negativity (112, 113) are among the macrophage-associated traits displayed by P388D1.

Originating from a spontaneous tumor in BALB/c mice, the PU5-1.8 cell line is a leukemia cell line that resembles macrophages. It has been extensively employed as an in vitro model for the investigation of monocyte differentiation, apoptosis, and cellular proliferation (114). PU5-1.8 cells are non-adherent and non-aggregating under conventional culture conditions (RPMI-1640 + 5% FBS + 1% penicillin/streptomycin; 5% CO₂; 37°C), which is compatible with undifferentiated macrophage characteristics. M-CSF induction can differentiate the cells (115).

WEHI-3, first reported by Warner et al. in 1969, is a granulocytic leukemia cell line with macrophage-like properties derived from the peripheral blood of tumor-induced BALB/c mice. It exhibits sustained secretion of IL-3, lysozyme, and granulocyte colony-stimulating activity (CSA), along with phagocytic capability and expression of complement C3 receptor (C3R) (116). In intervertebral disc degeneration research, the WEHI-3 model may simulate mechanisms where M1 macrophages exacerbate inflammation through IL-1β and IL-6 secretion, while M2 macrophages promote tissue repair via TGF-β production (117). In hepatocellular carcinoma studies, WEHI-3 mimics tumor-associated macrophages (TAMs) to investigate how M2 polarization supports tumor progression through IL-1β secretion and angiogenesis promotion (118). WEHI-3 proliferates under conventional culture conditions without exogenous M-CSF, in contrast to the majority of established macrophage-like cell lines that require M-CSF for proliferation (119). It most likely depends on self-secreted IL-3 and components of the basal medium (120, 121).

By transforming a hybrid strain of BALB/c and A.CA mice with the SV40 virus, the murine macrophage cell line BAC1.2F5 was created (119). It retains functions including Fc-mediated phagocytosis, Ia antigen expression, IL-1 secretion, and the synthesis of lysozyme, collagenase, and esterase, but it is entirely dependent on M-CSF for survival and proliferation, mimicking the behavior of primary macrophages (122). Securioside B’s growth-inhibitory action and capacity to trigger macrophage apoptosis through the mitochondrial pathway in the presence of L-cell-conditioned media (LCCM) were examined by Satoru et al. (123) in their investigation of the drug’s effects on BAC1.2F5.

Yet there is limited information on FC-1. In a 1983 study, FC-1 was compared to thioglycollate-induced peritoneal macrophages in the generation of plasminogen activator and six other macrophage cell lines (WEHI-3, J774A.1, RAW264, P388D1, PU5-R, and PU5-1.8). The findings indicated that FC-1 secretes non-plasminogen-dependent proteases in addition to plasminogen activators (124).

The bone marrow of BALB/c mice was utilized to create the murine monocyte/macrophage cell line LADMAC. It has poor adhesion and grows in suspension (125). The research of macrophage biology, in particular growth factor interactions and M-CSF-dependent mechanisms, has benefited greatly from the use of this cell line. In order to promote the survival and proliferation of BAC1.2F5, Wong et al. (125) showed that LADMAC cells release macrophage colony-stimulating factor (M-CSF), which can be employed in place of recombinant human CSF-1.

By transforming peritoneal macrophages from C57BL/6 mice with the SV40 virus, IC-21 was created (126). It maintains the phenotypic and functional characteristics of normal peritoneal macrophages, including phagocytic activity, lysozyme synthesis, particular receptors (TLR4, CD14, C3R), and antigen-presenting molecules (MHC-II). Because IC-21 is naturally adherent, it reduces experimental variability and does not require extra reagents like PMA (127). Nevertheless, IC-21 and primary peritoneal macrophages differ in the following ways: ①Enhanced erythrocyte-targeted phagocytosis is demonstrated by IC-21 (128). ②IC-21 has a more homogeneous tumor-binding capacity and binds tumor cells four times more efficiently than primary macrophages. Because of its easy cultivation and persistent, uniform tumor-binding characteristics, IC-21 is an advantageous model for understanding the molecular mechanisms behind activated macrophage subsets’ selective, high-affinity tumor contacts (129).

In 1952, the NCTC 1469 cell line was created using the liver tissue of healthy C3H/An mice. Studies on its chemotactic activity in the 1980s showed that this cell line, which resembles macrophages, responds consistently to different chemotactic substances (such as casein) in chemotaxis tests (130). Although NCTC 1469 CB, a mutant subline of NCTC 1469, lacks phagocytic activity toward erythrocytes coated with complement and IgG, it nonetheless possesses the functional and morphological characteristics of normal macrophages (131). Since more specialized macrophage models have been established, NCTC 1469 is now mostly used in hepatic research, such as investigations on liver regeneration, lipid metabolism regulation, and antioxidant capability (132–134).

SV40 viral transformation of alveolar macrophages collected from male BALB/c mice aged 7 weeks resulted in the establishment of the MH-S cell line (135). This cell line maintains several characteristic traits of alveolar macrophages, such as (1) typical macrophage morphology with adhesive properties; (2) phagocytic capacity; (3) enzymatic profiles that are esterase-positive and peroxidase-negative; (4) positive expression of surface membrane antigens Mac-1 and I-A; and (5) Fc receptor-dependent phagocytosis of sheep red blood cells (SRBCs) coated with immunoglobulin G. Additionally, LPS stimulation markedly increased constitutive IL-1 production.

ANA-1 is a macrophage cell line that is generated by immortalizing bone marrow cells from C57BL/6 (H-2b) mice through the J2 recombinant retrovirus, and it makes up the myeloid-monocytic lineage (136). These cells have dual adherent/suspended growth patterns, reveal morphology akin to that of macrophages, with prominent vacuolization and eccentric nuclei, and have high phagocytic activity against pathogens (e.g., Mycobacterium tuberculosis, Penicillium marneffei, and others) and particulate matter. ANA-1 expresses surface markers associated with macrophages, such as FcγR (Ly-17), Mac-1, and Ly-5, and may be involved in immune and inflammatory processes (137, 138).

In 1983, AMs from normal Sprague-Dawley (SD) rats collected through lung lavage served as a means to create the NR8383 cell line. NR8383 acquires an unlimited proliferative capability while maintaining normal macrophage activities through serial cloning and spontaneous immortalization (139). For in vitro research, especially in pulmonary inflammation, these semi-suspension cells, which have an oval or spherical shape, offer a consistent supply of highly sensitive alveolar macrophages (140). NR8383 expresses TGF-β precursor, upregulates its mRNA, and secretes pro-inflammatory cytokines (such as IL-1, TNF-β, and IL-6) when stimulated by bleomycin. The cells are extremely susceptible to endotoxins; 50% of proliferation is suppressed by 10 ng/mL LPS.

In 1998, the swine alveolar macrophages that gave rise to the pig alveolar macrophage cell line 3D4/21 were immortalized through SV40 transformation. It has persistent phagocytic capacity, nonspecific esterase activity, and morphology analogous to macrophages (141). Only a small proportion of 3D4/21 cells demonstrate the capacity to phagocytose latex beads following extended incubation (3 hours to overnight), as determined by Weingartl et al. (142). These results imply that while cytokine secretion functions are unaffected by immortalization, effective phagocytosis is somewhat lost.

MQ-NCSU and HD11 are immortalized cell lines of chicken macrophages. The avian myelocytomatosis virus MC29 strain has been applied to turn chicken bone marrow into the HD11 cell line, which has unmistakable macrophage-like characteristics. These include elevated generation of reactive oxygen species (ROS) and distinctive morphological characteristics (143). Derived from the spleen of chickens infected with the Marek’s disease virus, MQ-NCSU demonstrates mononuclear phagocyte lineage traits and malignant characteristics (109). According to Ahmed-Hassan et al. (144), LPS causes MQ-NCSU to undergo a type I interferon (IFN) response, which in turn causes the creation of nitric oxide (NO) and IFN-β.

ImKCs are isolated from hepatic tissue of male H-2Kb-tsA58 transgenic mice expressing the thermolabile mutant tsA58 of SV40 large T antigen and purified via F4/80 marker expression. These cells exhibit detectable p53 expression, elevated telomerase activity, and retained functional characteristics of primary KCs, with cytokine-induced polarization marker profiles comparable to primary counterparts. ImKCs may thus serve as functional substitutes for primary KCs in experimental systems (145).

In 1990, E. Braschi et al. created the BV2 cell line, a commonly used immortalized murine microglial model, by transfecting primary C57BL/6 mouse microglia with v-raf/v-myc oncogenes using a retroviral vector (146). BV2 cells lack peroxidase activity but have phagocytic and nonspecific esterase activity. In addition to constitutively secreting lysozyme, they also generate TNF and IL-1 when stimulated appropriately. Using MAC3 immunoreactivity, immunohistochemical profiling validates MAC1 and MAC2 expression (146).

In 1980, Tsuchiya et al. developed the THP-1 cell line from the peripheral blood of a kid with acute monocytic leukemia who was barely one year old. It is frequently employed in monocyte/macrophage studies and demonstrates the capacity to differentiate into different macrophage subtypes (19). Using retinoic acid (147), vitamin D₃ (148), or phorbol myristate acetate (PMA) (149), THP-1 cells can be separated into resting M0 macrophages based on metabolic and morphological similarities. Under certain triggers, these differentiated cells polarize into different subtypes and exhibit high expression of markers characteristic of macrophages, among them CD14 and CD68. For instance: M1 polarization: Pro-inflammatory cytokines (e.g., TNF-α and IL-6) are secreted by PMA-induced M0 macrophages stimulated with LPS and IFN-γ. M2 polarization: the production of anti-inflammatory cytokines, such as TGF-β and IL-10, is triggered by IL-4 and IL-13. However, since THP-1 lacks a defined differentiation strategy, PMA concentration must be optimized for certain experimental objectives (109).

The pleural effusion of a male patient with generalized diffuse histiocytic lymphoma was used by Sundstrom and Nilsson in 1974 to generate the U-937 cell line (111). U-937 is regarded as a relatively young monocytic population and exhibits monocyte-like characteristics. The following factors can cause it to terminally differentiate into macrophages: M0 macrophage differentiation is induced by PMA (150). Vitamin D₃: Encourages the development of monocytes into macrophages (151). Cytokine-driven polarization: M1 polarization is induced by IFN-γ, whereas M2 polarization is induced by IL-4 (152). U-937 allows for the functional diversity of macrophages in inflammatory and immunological responses thanks to these differentiation mechanisms.

The Seraphina Burkitt lymphoma cell line was transfected to produce the human B-cell precursor leukemia cell line BLaER1. The bone marrow of a female patient with acute lymphoblastic leukemia, trisomy 8, and chromosomal rearrangement t(1;19) served as the source of its progenitor line (153). Research shows that BLaER1 cells can be effectively reprogrammed to resemble macrophages, with a transcriptome similar to that of normal macrophages and improved adhesion, phagocytosis, and quiescent characteristics (154). When compared to other monocyte models with limited editing capabilities, BLaER1 offers improved genetic manipulability owing to its compatibility with CRISPR-Cas9 gene editing in the undifferentiated B-cell state (155–157).

The only documented human monocytic cell line that emerges from healthy peripheral blood is SC, which has been used in research pertaining to macrophages (158). Using PMA induction, a Chinese research team created a model of SC-derived macrophages. Among the main conclusions are (159): Phenotypic maturation: Compared to undifferentiated SC monocytes, SC macrophages exhibited a marked elevation of CD11b and CD14. Changes in morphology: LPS treatment resulted in the production of distinct pseudopodia and an elongated spindle-shaped morphology. M1 polarization is highlighted by increased secretion of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-8) and increased expression of M1 surface markers (CD80, CD86). These findings verify that SC macrophages replicate the functional and phenotypic characteristics of typical M1-polarized macrophages.

A 36-year-old Caucasian woman with acute myelomonocytic leukemia gave origin to the promyelocytic cell population characterized as the HL-60 cell line (160). Using substances like PMA (161) and vitamin D₃ (162), HL-60 cells can develop into macrophage-like cells, show phagocytic activity, and react to chemotactic stimuli. This cell line provides a special paradigm for examining how lipoprotein receptor expression and regulation are affected by macrophage development. Increased production of platelet-activating factors (PAF) is linked to HL-60 differentiation into macrophages (163). Further research showed that increased amounts of ethanolamine plasmalogens and phospholipids within arachidonic acid metabolism are correlated with macrophage-like differentiation in HL-60 (161).

Tardieu et al. (1995) immortalized human fetal brain-derived primary microglial cultures SV40-dependently to create the HMC3 cell line (164). Although transformed HMC3 cells retain important primary microglia physical and phenotypic characteristics, they proliferate more quickly. The astrocytic marker GFAP is negative in resting HMC3 cells, but they show significant positivity for the microglial/macrophage markers IBA1, CD68, and CD11b. When HMC3 cells are at rest, the activated microglia marker MHC-II is not visible; nevertheless, after IFN-γ treatment (10 ng/mL, 24 h), it is markedly elevated (56, 164).

Three primary murine macrophages—SMs, PMs, and BMDMs—were compared in terms of their properties by Zhao et al. (33). The study found that 48 hours of LPS+IFN-γ stimulation could polarize all three types of macrophages into an M1 state, while 48 hours of IL-4 stimulation could polarize them into an M2 state. While there were no appreciable variations in M2-polarizing capacity, SMs showed a greater M1-polarizing capability than the others. The most homogeneous macrophages are produced via adherent culture of BMDMs; however, their phenotype leans more toward M2. It was determined that although BMDMs offer a large number of uniform macrophages, TRMs cannot be completely replaced by them. IPSDMs showed a considerable but lesser bacterial phagocytic ability than PBMCs, according to Monkley et al. (171), while their transcriptome profiles and pro-inflammatory responses were similar. Conflicting research, however, indicates that IPSDMs tend to have an M2 phenotype, which lowers phagocytic activity (172, 173).

In order to assess how J774A.1, WEHI-3, P388D1, IC-21, NCTC 1469, and U937 responded to several chemotactic agents (casein, an N-formyl tetrapeptide, and culture supernatants of murine SL2 lymphoma cells), Terheggen et al. (130) performed chemotactic activity experiments on these cells. The findings showed that the cell lines’ susceptibility to various chemoattractants varied significantly. The most potent chemotactic activity was demonstrated by J774A.1 and WEHI-3 macrophage-like cells toward casein and N-formyl tetrapeptide, respectively. Van et al. (174) examined the levels of surface antigen expression, M-CSF secretion, and peroxidase activity in five cell lines: NCTC 1469, J774A.1, WEHI-3, IC21, and P388D1. According to the study, M-CSF secretion was unique to WEHI-3 cells, whereas peroxidase activity was present in all five cell lines. M1/69 and Mac-1 antigens were significantly expressed by P388D1 cells, while they were faintly expressed by NCTC 1469 and IC21 cells. J774A.1 cells showed the reverse pattern, with strong M1/69 and low Mac-1 expression in WEHI-3 cells. Nibbering et al. (175) compared quantitative data from four cell lines (WEHI-3, P388D1, J774A.1, and PU5-1.8) on surface antigen expression (e.g., F4/80, CDb11) with data from different mononuclear phagocytes. The four macrophage-like cell lines showed clear phenotypic differences, with WEHI-3 and P388D1 exhibiting the highest association with monocytes, according to the data. None of the macrophage-like cell lines closely matched any particular mononuclear phagocyte population, even though they shared several characteristics with mature mononuclear phagocytes. In comparison to RAW 264.7 and J774A.1 cells, IC-21 cells had a higher degree of differentiation than P388D1 cells (176) and more filopodia and membrane ruffles (176, 177).

The phagocytic activity and polarization capacity of THP-1 and PBMCs were contrasted by Shiratori et al. (152). According to the study, THP-1 has higher phagocytic activity and a stronger propensity toward M1 polarization than PBMCs. IPSDMs exhibit more distinctive macrophage surface characteristics and express larger amounts of CD86 and MRC1 than U-937 (178). The immunobiological profile of BMDMs can only be partially replicated by THP-1 cells, according to Gaidt et al. (179). Using four macrophage cell lines (J774A.1, PU5-1.8, WEHI-3, and RAW 264.1) and newly obtained primary macrophages from C3H/He mice under standard culture conditions, Nibbering et al. (180) examined the production of N-nitrosamine during immunological stimulation in vitro. The findings showed that when LPS was stimulated, all cell types produced nitrite and N-nitrosomorpholine, and that IFN-γ amplified this action. According to Bodel et al. (181), J774A.1, PU5-1.8, P388D1, and WEHI-3 were still able to manufacture lysozyme and pyrogens on their own in vitro. Via et al. (182) assessed the ability of two human macrophage/monocyte cell lines and four murine macrophage cell lines to degrade unmodified LDL (native LDL) and acetylated low-density lipoprotein (AcLDL). According to the findings, PU5-1.8, HL-60, and U-937 are not appropriate models for researching scavenger pathways; however, P388D1, J774A.1, and RAW 264.7 are. The morphology and quantitative surface expression of C3b antibodies in primary macrophages, J774A.1, PU5-1.8, WEHI-3, and P388D1 were compared by Furth et al. (183). According to morphological data, J774A.1 and WEHI-3 cell lines were almost the same in most ways, while P388D1 was the most different from them but most comparable to primary macrophages. Furthermore, WEHI-3 and P388D1 cell lines exhibited significantly reduced surface C3b receptor expression in comparison to the other two cell lines.

Plasmid DNA, siRNA, and/or shRNA transfection has been portrayed to be possible in RAW264.7 cells, but it is very difficult in primary macrophages, where it commonly results in cellular death (184–187). Interestingly, J774.1 cells share functional similarities with primary macrophages, yet exhibit distinct responses from RAW264.7 cells: transfection with plasmid DNA induces significant cell death, whereas mRNA transfection maintains cellular viability (188). For eukaryotic cells, common transfection methods include electroporation, mechanical methods (such as gene guns), and viral vectors. Yet, since almost all known transfection processes severely impair cellular survival or obstruct their differentiation and polarization tendencies, macrophages are especially resistant to transfection techniques (188). In order to overcome these constraints, optimal transfection techniques have been suggested: Michael et al. (189) created a modified nucleofection-based method that effectively transfects THP-1 cells and allows them to differentiate into mature macrophages while maintaining cellular viability and functionality. Similarly, using an improved electroporation-based, non-viral nucleofection technique that preserved cellular integrity and physiological functioning, Maeß et al. (190) successfully transfected human THP-1 macrophages. Moreover, recent studies demonstrate that engineered nanoparticles (191) and the macrophage-specific editing (MAGE) system (192) enable highly efficient transfection of plasmid DNA/RNA in primary macrophages, surpassing conventional approaches in both delivery efficacy and specificity ( Tables 1 , 2 ).