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Review

Application of Conditioned Medium for In Vitro Modeling and Repair of Respiratory Tissue

by
Fairuz Izan Muhammad Firdaus
1,
Ab. Karim Nashihah
1,
Mh. Busra Mohd Fauzi
1,
Maarof Manira
1,
Saim Aminuddin
2,3 and
Yogeswaran Lokanathan
1,*
1
Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur 56000, Malaysia
2
Graduate School of Medicine, KPJ Healthcare University College, Kota Seriemas, Nilai 71800, Malaysia
3
KPJ Ampang Puteri Specialist Hospital, Ampang 68000, Malaysia
*
Author to whom correspondence should be addressed.
Appl. Sci. 2023, 13(10), 5862; https://doi.org/10.3390/app13105862
Submission received: 3 March 2023 / Revised: 3 May 2023 / Accepted: 5 May 2023 / Published: 10 May 2023
(This article belongs to the Special Issue Advanced Stem Cell Technology and Regenerative Medicine)
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Abstract

:
Background: The idea of exploring respiratory therapy in vitro predominantly guided by cell-secreted substances has gained ground in recent years. A conditioned medium (CM) consists of protein milieu that contains a diverse spectrum of cytokines, chemokines, angiogenic agents, and growth factors. This review evaluated the efficacy of using CM collected in an in vitro respiratory epithelial model. Methods: Twenty-six papers were included in this review: twenty-one cellular response studies on respiratory secretome application and five studies involving animal research. Results: The CM produced by differentiated cells from respiratory and non-respiratory systems, such as mesenchymal stem cells (MSC), exhibited the similar overall effect of improving proliferation and regeneration. Not only could differentiated cells from respiratory tissues increase proliferation, migration, and attachment, but the CM was also able to protect the respiratory epithelium against cytotoxicity. Most non-respiratory tissue CM was used as a treatment model to determine the effects of the therapy, while only one study used particle-based CM and reported decreased epithelial cell tight junctions, which harmed the epithelial barrier. Conclusion: As it resolves the challenges related to cell development and wound healing while simultaneously generally reducing the danger of immunological compatibility and tumorigenicity, CM might be a potential regenerative therapy in numerous respiratory illnesses. However, additional research is required to justify using CM in respiratory epithelium clinical practice.

1. Introduction

Respiratory illnesses such as chronic obstructive pulmonary disease (COPD), asthma, pulmonary hypertension, and occupational lung diseases are the third largest cause of mortality worldwide, affecting millions each year [1]. Acute and chronic lung disorders, including bronchial asthma, pneumonia, and COPD, depend on the interaction of airway epithelial cells (EC) with pathogens and endogenous signals [2]. The current treatments are ineffective, and some treatments are simply palliative, as they treat the symptoms but are ineffective for rescuing or regenerating cellular function or even halting the degenerative process [3,4]. From the nasal cavity to the bronchi, the respiratory tree is lined with pseudostratified columnar ciliated epithelium. As the respiratory epithelium is in constant contact with the external environment, which includes germs, allergens, and air pollutants [5,6], the fundamental task of protecting the airway integrity is in constant jeopardy, resulting in epithelial degradation and the development of acute and chronic lung illnesses [7,8,9].
Given that the respiratory system is the primary pathway for airborne toxicants, the respiratory epithelium presents a physical barrier with tight junction (TJ) complexes that are in immediate contact with the external environment and protect the underlying tissue. In humans, different cell types, including mucus-producing goblet cells, ciliated cells, and non-ciliated cells, are arranged into a protective pseudostratified epithelium, depending on the airway segment [10,11]. Lung lining fluid, which consists of mucus in the upper airways and surfactant in the alveoli, protects the epithelia that line the respiratory tract through mechanical, antibacterial, and antioxidant activities [12,13]. As the airways are continually exposed to foreign agents and infections, airborne toxicants can potentially translocate through an intact or compromised epithelial barrier, generating possible systemic effects [12,14,15]. The epithelial damage can even result in the loss of surface epithelial integrity and partial or total epithelium shedding [16,17]. Consequently, developing and validating in vitro models to assess the biological responses to disruptions of this first-line upper airway defense is critical [18,19,20].
Tissue engineering models strive to mimic the natural components and interactions of tissues by utilizing biological and biochemical advances to achieve the closest feasible physiological similarity [21]. Understanding cellular activity at the molecular level is therefore critical to preserving the cell quality and regenerative potential to produce better-quality epithelium for in vitro uses. Tissue alternatives, such as 3D cell cultures and organoids, are being developed for a range of fundamental and practical reasons, including therapeutic applications and in vitro testing, and the development of human cell- and tissue-based in vitro screening methods has accelerated [22,23,24]. However, the use of cells is accompanied by disadvantages that include dedifferentiation after implantation, the need for in vitro expansion to obtain more cells for in vivo regeneration, and donor site morbidity due to cell isolation, particularly in patients debilitated by age and disease. Furthermore, cell proliferation and regeneration capacity decrease as people age and illness severity increases [25,26,27,28]. While preclinical studies are widely used in vitro models, their lack of physiological complexity, especially in epithelial models, renders them unsuitable for toxicity research and impedes the effective creation of new therapeutic medicines [29,30,31,32]. Thus, despite their prevalent use, such models are nearing the limits of their ability to satisfy modern drug research demands [29].
A cell-free approach that uses only cell-produced soluble components, the secretome or conditioned medium is needed to alleviate the limitation of both preclinical and clinical testing, particularly in in vitro models [25]. A collection of cytokines, chemokines, angiogenic factors, and growth factors, the CM is increasingly recognized for its function in controlling various physiological processes [33]. Many of these secretory proteins have biological roles that include anti-inflammatory, antifibrotic, and immunomodulatory properties [34,35,36]. Therefore, the importance of the secretome in tissue regeneration has been recognized. A recent study reported that airway epithelium-derived secreted factors enhance early airway epithelial repair by decreasing inflammation. This is accomplished by controlling factor secretion by the airway epithelium, highlighting the varied biological capabilities of the cell type [37,38]. Moreover, the CM has demonstrated significant advantages over cell-based in vitro modeling for preclinical and clinical testing, as the CM has the potential to overcome the cancer development risk caused by improper cell grafting and can be used with off-the-shelf materials that do not require isolating the patient’s stem cells. This has resulted in significant advantages for preclinical and clinical research, rendering the CM a better and more effective choice [39,40]. Given the aforementioned information, the aim of this review was to evaluate the effectiveness of CM application in epithelium in vitro models. Furthermore, preclinical experiments that used cell factor-rich CM for respiratory regeneration using a cell-free approach were evaluated.

2. Materials and Methods

2.1. Search Strategy

The literature was evaluated comprehensively to identify the relevant research describing secretome characterization and in vitro experiments to assess secretome use in pulmonary tissue healing. This descriptive literature review was conducted using three databases over a period of a year: PubMed (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA), Scopus (Elsevier, Amsterdam, The Netherlands), and ISI Web of Science (WoS, Clarivate Analytics, Philadelphia, PA, USA). A systematic search was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Relevant articles from the previous 10 years (from January 2012 to February 2022) were searched. The keywords were (“Respiratory” (MeSH) OR “Airway” (MeSH) OR “Lung” (MeSH) OR “Epithelial” (MeSH) OR “Epithelium” (MeSH)) AND (“Secretome” (MeSH) OR “Conditioned medium” (MeSH)) AND (“Culture” (MeSH) OR “Model” (MeSH) OR “In vitro” (MeSH) OR “In vitro model” (MeSH) OR “In vitro co-culture” (MeSH)). We identified studies where both 2D and 3D in vitro models were used in respiratory studies. Additional studies not found in the initial search were identified by analyzing the reference lists from the included articles. Duplicate articles were deleted with a reference manager (www.mendeley.com) (accessed on 15 August 2022).

2.2. Inclusion Criteria

Due to a lack of translation resources, only English articles were included in this review. Original research papers that explored the influence of a secretome in an in vitro model with a focus on respiratory tissue were included. This review covered studies that used the CM from non-respiratory tissue in conjunction with the presence of respiratory cells. Studies on wound healing cells and disease models, including macrophages and mesenchymal cells, were also included.

2.3. Exclusion Criteria

All secondary literature and original publications that included only in vivo and clinical stage research, articles authored or submitted in languages other than English, and gene expression studies from unrelated cells to demonstrate the applications and effects of secretome usage were excluded.

2.4. Data Extraction and Management

In this review, the articles were reviewed in three stages. The initial stage was to screen titles and delete those that did not meet the inclusion requirements. The second stage was to evaluate the remaining abstracts for unsuitable articles based on the inclusion criteria. Following full-text examination by two independent reviewers, papers that did not match the inclusion criteria were removed.

3. Results

3.1. Search Results

A total of 432 articles were identified as potentially relevant. The initial screening removed 243 publications, as they were non-original articles, not authored or submitted in English, duplicates, or had a title or abstract that did not match the inclusion standards. The reviewers studied the complete texts of the remaining 189 papers, of which 161 were eliminated, as they did not meet the inclusion requirements. After selection, a total of 26 papers were chosen for evaluation. Figure 1 depicts a flowchart of the article selection process.

3.2. Study Characteristics

All selected publications contained a parameter to describe the biochemical features of the secretome, from specific marker expression changes to morphological changes. The article discusses 26 studies that were conducted on cellular response research from respiratory secretome application. Out of these 26 studies, 21 focused on this particular area of research. The non-respiratory tissues used in the in vitro experiments are listed in Section 3.4 of this article. It is important to note that, although there were five studies conducted on animals, their data were not included in the analysis due to the exclusion criteria specified in Section 2.3 of this article. The article classification divided the data into two categories of cells used for secretome collection: (1) respiratory system cells or tissues that mainly consisted of primary cells and cell lines such as primary respiratory EC (REC) and lung carcinoma EC (A549 cells) and (2) cells or tissues other than respiratory, such as mesenchymal stem cells (MSC) and macrophages. The articles in both categories were published between January 2012 and February 2022.

3.3. Application of CM from Respiratory Cells or Tissues

Primary cells or cell lines were one of the most commonly used tissues for secretome collection, and they were involved in 10 of the 26 studies reviewed. In addition to such cells, disease-related tissues such as lymphangioleiomyomatosis (LAM) cells were also investigated as a disease model [41]. The result metrics used included cell viability assays, proliferation assays, scratch wound migration assays, immunofluorescence, Western blotting, and enzyme-linked immunosorbent assays (ELISA). While most studies examined both proliferation and migration assays, some found that a faster migration rate was often associated with a greater impact of tissue-conditioned media on cell proliferation [37,42,43]. Four studies examined cell viability assays [41,44,45,46], where a higher proportion of viable cells was accompanied by a larger estimation of the number of live cells. The 10 studies also conducted specific marker expression experiments, namely immunofluorescence assays, PCR, ELISA, and Western blotting. CK18, KI67, and MUC5B were among the genes expressed [42]. Table 1 summarizes where cell-released factors from respiratory disorder-related tissue, such as LAM-associated fibroblasts and idiopathic pulmonary fibrosis fibroblasts, demonstrated positive effects on respiratory regeneration in vitro, including increased proliferation, migration, and protection against apoptosis. However, cancer-associated fibroblast CM was found to accelerate lung cancer cell metastasis.

3.4. Application of CM from Non-Respiratory Cells or Tissues

Non-respiratory tissues were also included to elucidate the role of the secretome in in vitro modeling to discover secretome applications in respiratory in vitro models. Adipose-derived stem cells [51], THP-1 monocytes [52,53], 3T3 cells [54], MSC [36,55,56,57,58,59,60,61], and macrophages [62] were among the cells used. Particle-exposed cells such as Buenos Aires urban air particles (UAP-BA)- and residual oil fly ash (ROFA)-treated cells [63] were also included, along with bacteria secretome such as that of Staphylococcus aureus [64]. A total of 16 articles were evaluated, which involved >20 separate tests. The most common tests, which were performed in seven studies, were viability and migration assays [36,51,52,54,55,56,62]. Aside from specific marker expression assays, such as immunofluorescence assays, PCR, ELISA, and Western blotting that were evaluated in 15 of the 16 publications, some studies performed uncommon tests, such as ERK1/2 phosphorylation [57], a PPAR activity assay [62], Hoechst 33258 staining assay [58], and luciferase assay [53], all of which were conducted in a single study. Table 2 summarizes the results of various studies evaluating conditioned media (CM) from different cell types and bacteria, which demonstrated their ability to promote regeneration, exert protective effects, inhibit fibrosis, and induce epithelial breakdown. Notably, the CM from MSC showed the most promising results in promoting proliferation, wound closure, and exerting antifibrotic effects.

4. Discussion

In this study, we collected all available data in the literature and critically examined whether CM derived from various types of differentiated and non-differentiated cells greatly influenced and aided in vitro respiratory regeneration. We generated an objective and unambiguous assessment of the published scientific evidence that resulted in a review conducted specifically to evaluate the effect of CM on respiratory regeneration. However, our results are hampered by possible bias. Only in vitro research was used as an inclusion criterion, resulting in limited findings. This requirement is justified by the fact that the study goal was to clarify the significance of secretome application on in vitro models. Second, non-English literature was excluded due to a lack of translation capability. Furthermore, we did not examine grey literature publications. In our review, we aimed to eliminate duplicate publications. For future evaluations, statistical analysis is proposed to improve the outcome of this study. Nevertheless, the use of secretomes during in vitro investigations should provide solid molecular support when conducting higher-degree studies.
The last 10 years of research has supported the concept that cell-released factors have a therapeutic effect in vitro on various respiratory illnesses and physiological changes. Despite the recent breakthroughs in in vitro modeling, there remain gaps in replicating the physiological complexity of native cells. Thus, researchers have extensively examined the biochemical impact of using the secretome to imitate extracellular matrices to achieve the same microphysiological effects as native cells [66,67,68,69]. The use of the secretome might potentially avoid the concerns regarding immunological compatibility, tumorigenicity, and infection transmission associated with cell treatments. Secretome usage might also significantly minimize the time and expense involved in cell proliferation and maintenance.
In vitro research has investigated the effects of CM from differentiated cells on respiratory epithelium tissues. The findings suggested that CM may be used successfully to improve the functioning of both healthy and diseased cells [42,46,50]. CM derived from primary cells such as nasal fibroblasts enhance cell proliferation, attachment, and migration [42,46] and also exert a cytoprotective effect on REC [42]. The CM from cell lines such as A549, when cocultured with THP-1, aids in the reduction of cancer cell metastasis [50].
Preclinical studies from this study supported the idea that cell-released factors from respiratory disorder-related tissues in vitro may exert a therapeutic effect on respiratory regeneration. In vitro data demonstrated that LAM-associated fibroblast (LAF) CM increased the pace of epithelial healing by increasing cell proliferation, migration, and the capacity to protect against apoptosis [41]. CM from idiopathic pulmonary fibrosis (IPF) fibroblasts improved the restoration ability with changes in the form of higher amounts of matricellular protein, especially SPARC [47], while cancer-associated fibroblast (CAF)-CM accelerated lung cancer cell metastasis by increasing the proliferative and invasive capacities [49].
Factors released by other tissue groups, such as MSC, were also studied primarily for their ability to induce regeneration in respiratory damage. MSC-CM promoted proliferation and wound closure in respiratory cells [36,56,57,60,61], protected the cells against apoptosis [58], and exerted antifibrotic effects [55]. Little is still known about the CM from other cell types, such as adipose-derived stem cells [51], THP-1 monocytes [52,53], 3T3 cells [54], and macrophages [62]. However, the CM from these cells exerted positive effects, such as a protective ability against EMT in lung cancer [51,62]. Some CM were also used in treatment studies, for example, a study of the effect of esculetin treatment of EC induced by monocyte-derived macrophages determined that it inhibited lung fibrosis and epithelial breakdown caused by the macrophages [52]. Some studies used CM from bacteria, specifically S. aureus [64], to explore the EC barrier function, which resulted in decreased cell TJ. In that study, the CM was derived from UAP-BA and ROFA particles rather than cells, and the factors produced by the particles reduced cell survival and caused inflammation [63].
Although the CM from respiratory tissues has demonstrated beneficial effects on respiratory cells, the effects of CM from non-respiratory tissues can differ significantly. Studies have indicated that CM from non-respiratory tissues, such as MSCs, may lead to immunomodulatory effects that can help reduce inflammation and exert antifibrotic effects on respiratory cells [36,55,56,57,58,59,60,61], while the CM from adipose tissue can increase inflammation and oxidative stress [51]. Consequently, it is crucial to thoroughly evaluate the origin of CM and its potential consequences prior to employing it for therapeutic purposes. The dissimilarities observed in the impact of CM from respiratory and non-respiratory tissues could be attributed to variations in the molecular makeup of the CM and the distinct physiological characteristics of the cells responsible for its secretion [70].
The molecular mechanisms and key protein factors involved in the therapeutic actions of the secretome have received attention. Nevertheless, they have not been fully explored, despite the importance of secretome research, which requires wide characterization before clinical research can be conducted. In some in vitro studies related to secretome applications from respiratory diseases, IL-6 expression became the protein of focus rather than H2AXγ, p21, TNF-α, and IL-8. This was because the secretome from unhealthy tissue was induced to the epithelium and altered IL-6 production, which is involved in activating EMT and metastasis [45,48,49,50]. IL-6 stimulates the IL-6 receptor (IL-6R), which initiates signaling via the Janus kinase–signal transducers and activators of transcription (JAK–STAT) signaling pathway and the NF-κB signaling pathway [71], hence triggering an inflammatory reaction and activating EMT processes. Figure 2 provides a detailed overview of the mechanism of action of IL-6.
An in vitro study reported EGFR activation induced by activation of the ERK1/2 signaling cascade present in the secretome, which aided wound healing to the epithelium [57]. Another study reported that AKT and PI3K signaling were involved in secretome application in vitro, as they induced a Na+ influx through the epithelium sodium channel (ENaC) activity [59]. Contrastingly, only one in vitro study used a bacterial secretome (S. aureus), which involved ZO-1 expression [64]. The ZO-1 protein aids cell adhesion and TJ management; hence, the secretome created ZO-1 expression discontinuity and damaged the respiratory epithelial TJ [64]. IL-8 expression was the focus in terms of particle-CM, as the IL-8 protein was increased due to the cytotoxic effect of the proinflammatory cytokines when the epithelium was exposed to the CM [63]. A comprehensive summary of the findings in this study is presented in Figure 3.
Recent research has also demonstrated the use of exosomes rather than secretomes for in vitro applications. Although both secretions have received attention for their therapeutic potential in respiratory regeneration, they focus on two separate aspects. While the secretome mainly consists of messenger chemicals discharged to the outside, including microvesicles and exosomes, both secretomes and exosomes confer distinct benefits and target various metabolic pathways and affect the respiratory epithelium. The secretome was occasionally linked to the immunological and endocrine system pathways and protein processing in the endoplasmic reticulum. Contrastingly, exosome-specific proteins were mostly related to endocytosis, cell junctions, platelet activation, and other cell signaling pathways [73]. In contrast to the highly complex nature of the secretome, exosomes can be engineered to carry a specific cargo based on their selectivity for parent cell types [74], enabling targeted delivery to specific cells or tissues and leading to improved therapeutic outcomes with a greater specificity and reduced risk of adverse effects, including potential protumor effects [75,76]. Moreover, exosomes show a greater stability compared to secretomes because of their protective lipid bilayer, which shields them from enzymes and other agents that can degrade proteins and other biomolecules [77,78]. This property makes exosomes more suitable for clinical applications, particularly as therapeutic targets. Furthermore, the limitations of advancements for this study, such as sample and batch deviations worldwide, should be addressed to clarify secretome studies [79]. The biochemical properties of the secretome were shown to be heterogeneous, which makes identifying particular components and developing therapeutic approaches difficult. Further research is needed to elucidate the biological functions and potential therapeutic applications of the diverse secretome components. Researchers should also discuss the results and how they can be interpreted from the perspective of previous studies and their working hypotheses. The findings and their implications should be discussed in the broadest context possible. Future research directions may also be highlighted.

5. Conclusions

The CM of differentiated cells from respiratory and non-respiratory tissues might be used as a biological therapy in various respiratory diseases. Using CM can address several challenges associated with using cells, including safety concerns, high costs, immune rejection from using allogenic cells, quality management, tumor formation risk, and the limited survival of implanted cells at the injury site during clinical trials. While preclinical research has been conducted throughout this decade, clinical trials on human models are currently ongoing to improve the respiratory regeneration process and render it a safe and successful medicine. Guidelines to standardize the evaluation of new tissues used in secretome application for respiratory regeneration in vitro are lacking. Based on the papers included in this review and a comparison of the study outcome measures with the accessible clinical outcomes, the MSC-CM has emerged as a promising biological therapy for respiratory diseases, addressing several challenges associated with using cells. It is proposed that more clinical trials on secretome applications be prioritized rather than in vitro models to prove the efficacy of using this protein milieu.

Author Contributions

Conceptualization, Y.L.; Data curation, F.I.M.F.; Formal analysis, F.I.M.F.; Funding acquisition, S.A. and Y.L.; Investigation, F.I.M.F.; Resources, S.A. and Y.L.; Supervision, M.B.M.F., S.A., M.M. and Y.L.; Writing—original draft, F.I.M.F.; and Writing—review and editing, A.K.N., M.B.M.F., S.A., M.M. and Y.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by grants provided by the Ministry of Higher Education (MOHE) Fundamental Research Grant Scheme, Malaysia: (FRGS/1/2020/SKK0/UKM/02/7).

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Universiti Kebangsaan Malaysia Research Ethics Committee (FRGS/1/2020/SKK0/UKM/02/7; date of approval: 7 December 2021).

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

This article was written under a project that was approved by the Universiti Kebangsaan Malaysia Research Ethics Committee and supported by the Ministry of Higher Education (MOHE) Fundamental Research Grant, Scheme, Malaysia: (FRGS) FRGS/1/2020/SKK0/UKM/02/7.

Conflicts of Interest

The authors declare no conflict of interest.

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Applsci 13 05862 g001 550
Figure 1. PRISMA flow diagram.
Figure 1. PRISMA flow diagram.
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Figure 2. Depicts the role of IL-6 in activating the JAK-STAT pathway, which is responsible for many of the observed therapeutic effects of CM on respiratory tissues [72]. IL-6 binding to its receptor induces the activation of the JAK-STAT signaling pathway, which, in turn, leads to the phosphorylation of STAT proteins and their subsequent dimerization, nuclear translocation, and regulation of gene transcription, including inflammatory cytokines. Data from Zhou et al. (2022) [72].
Figure 2. Depicts the role of IL-6 in activating the JAK-STAT pathway, which is responsible for many of the observed therapeutic effects of CM on respiratory tissues [72]. IL-6 binding to its receptor induces the activation of the JAK-STAT signaling pathway, which, in turn, leads to the phosphorylation of STAT proteins and their subsequent dimerization, nuclear translocation, and regulation of gene transcription, including inflammatory cytokines. Data from Zhou et al. (2022) [72].
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Figure 3. Illustrates the effects of CM application on respiratory tissues, highlighting the key protein factors involved in the observed effects. The therapeutic actions of the secretome involve various molecular mechanisms and key proteins, including IL-6, EGFR, AKT, PI3K, and ZO-1, which affect wound healing, EMT, and respiratory epithelial TJ, as well as proinflammatory cytokine expression.
Figure 3. Illustrates the effects of CM application on respiratory tissues, highlighting the key protein factors involved in the observed effects. The therapeutic actions of the secretome involve various molecular mechanisms and key proteins, including IL-6, EGFR, AKT, PI3K, and ZO-1, which affect wound healing, EMT, and respiratory epithelial TJ, as well as proinflammatory cytokine expression.
Applsci 13 05862 g003
Table
Table 1. Applications of CM from respiratory cells or tissues.
Table 1. Applications of CM from respiratory cells or tissues.
CELL TYPECM CHARACTERISTICSEXPERIMENTAL EVALUATIONRESULTS AND CONCLUSIONS
Lung carcinoma EC (A549), pulmonary alveolar type 2 cells (AT2), tuberous sclerosis complex 2 cells (TSC2), LAM cells [41]From LAM-associated fibroblasts (LAF) cultured for 24 hImmunohistochemistry, fluorescein diacetate viability assay, immunofluorescence, proliferation assay, Boyden chamber migration assay, scratch wound migration assay, caspase 3/7 activity assay, ELISA
   -
Nodular AT2 expressed protein associated with EC activation, such as matrix MMP7 and FGF7.
   -
TSC2 + LAF derived from LAM exhibited ↑ FGF7 compared to LAF alone.
   -
LAF-CM + A549 exhibited positively chemotactic feature, ↑ rate of repair and migration, and protected against apoptosis.
   -
LAF-CM accumulation in LAM may alter AT2 cell distribution and proliferation. FGF7 mediated crosstalk between EC and LAF. Thus, this could present new therapeutic options for LAM.
Primary human lung cells, lung carcinoma EC (A549), human distal lung EC (NCI-H441) [44]From lung-derived mesenchymal stromal cells (LMSC) cultured for 24 hCell viability, electrical resistance and electric field injury, scratch wound, qPCR
   -
LMSC-CM ↓ number of necrotic cells on H2O2-induced cells that caused cellular stress, while also ↑ number of viable cells.
   -
LMSC-CM ↑ in initial recovery phase of cells from electric field-induced injury that caused detachment.
   -
LMSC-CM ↑ in monolayer recovery, accelerating wound repair from scratch injury in A549 cells.
   -
Despite no significant difference between COPD and non-COPD control-derived LMSC, endogenous LMSC were nevertheless identified as a potential target for treatment techniques aimed at correcting aberrant lung tissue repair in COPD.
Lung tissue from idiopathic pulmonary fibrosis (IPF), primary lung fibroblasts (LF). [45]From lung carcinoma EC (A549), LF derived from IPF (IPF-LF), and senescent-induced LF cultured for 72 hSenescence induction protocol, cell viability, protein immunoblotting, ELISA, immunofluorescence staining, PCR
   -
Exposure of non-senescent control LF to CM from IPF-LF, senescent-induced LF, and AEC ↑ senescence markers, including H2AXγ, p21, IL-6, and IL-8.
   -
Senescent fibroblast CM inhibited fibroblast growth.
   -
“Senescent-induced senescence” events may occur in human LF in vitro primary culture and could be the source of an abnormal number of senescent cells in the lungs of IPF patients.
Human distal lung EC (NCI-H441), primary human type II alveolar EC (AEC) [47]From IPF fibroblasts (IPFF) and normal human LF (NHLF) cultured for 48 hMacromolecular permeability assay, cell counting, mass spectrometry, scratch wound healing assay, Luminex multiple assays, cell transfection, western blotting, immunofluorescence staining
   -
IPFF-CM ↑ migration rate in wound repair response compared to NHLF-CM on AEC.
   -
↑ level of matricellular protein SPARC in IPFF-CM compared to NHLF-CM; no difference for MMP2 protein.
   -
SPARC also ↑ in IPFF compared to AEC and H441 cells.
   -
SPARC secretion ↑ alveolar epithelial chronic activation via an integrin–focal adhesion–cellular junction axis, resulting in epithelial barrier collapse and increased macromolecular permeability.
   -
SPARC is an important mediator of epithelial–mesenchymal paracrine signaling and hence could be the reason for the increased restoration ability of normal EC and might become a potential therapeutic target to promote restoration in IPF patients.
REC [46]From nasal fibroblasts (NFCM) in defined keratinocyte medium (NFCM_DKSFM) and in F-12 and Dulbecco’s modified Eagle’s medium (NFCM_FD) cultured for 72 hCell viability, qPCR, scratch assay, wound healing assay
   -
NFCM_DKFSF ↑ cell attachment compared to NFCM_FD and DKSFM medium alone.
   -
NFCM_DKFSF ↑ migration rate compared to NFCM_FD and DKSFM.
   -
DKSFM ↑ growth rate compared to NFCM_DKSF.
   -
Although NFCM_DKFSF and NFCM_FD did not exhibit much difference, NFCM provided optimal culture conditions for REC through cell attachment and migration.
Human bronchiole EC (HBEC), lung carcinoma EC (A549), human lung adenocarcinoma cells (SPC-α-1) [48]From non-small cell lung cancer (NSCLC) and human mast cells (HMC) cultured for 24 hRT-PCR, ELISA, immunofluorescence, western blotting
   -
HMC infiltrating into NSCLC ↑ compared to infiltrate into normal tissues.
   -
NSCLC released CCR5 protein that could cause HMC recruitment into tumor microenvironment.
   -
HMC-CM ↑ A549 and SPC-α-1 migration and EMT.
   -
↑ protein level of IL-6, TNF-α, and especially IL-8, in HMC-CM suggested that IL-8 is the main factor causing EMT and migration.
   -
Inhibiting IL-8 reversed EMT and NSCLC cell migration.
Lung carcinoma EC (A549), human squamous cell carcinoma (SK-MES-1), human lung large cell carcinoma cells (H661) [49]From cancer-associated fibroblasts (CAF) and normal fibroblasts (NF) cultured for 72 hCell proliferation assay, wound healing assay, cell invasion assay, qPCR, western blotting, ELISA, immunofluorescence
   -
CAF-CM ↑ cancer cell proliferation compared to NF-CM and control.
   -
CAF-CM ↑ EMT and metastatic potential due to ↑ E-cadherin, vimentin, VEGF, and MMP2 expression.
   -
CAF-CM ↑ invasion ability compared to NF-CM and control.
   -
CAF-CM and NF-CM ↑ IL-6 levels than in control, demonstrating that IL-6 could induce EMT and enhance metastasis potential.
   -
Inhibiting IL-6 prevented CAF-CM-induced EMT and lung cancer cell migration.
Lung carcinoma EC (A549) [50]From THP-1 cells co-cultured with A549, treated with tectorigenin (T-CM) and untreated co-culture (UT-CM) for 24 hWound healing assay, invasion assay, immunoblotting
   -
A549 activated pro-inflammatory response of THP-1 cells by ↑ TNF-α and IL-6 production.
   -
Tectorigenin inhibited TNF-α and IL-6 released by THP-1 cells.
   -
T-CM inhibited cell migration rate and cell invasion compared to UT-CM.
   -
T-CM ↓ EMT, observed by ↓ E-cadherin and Snail expression.
   -
Tectorigenin can prevent lung cancer by inducing an inflammatory and pro-metastatic response in monocytes.
Hepatoma cell line (Bel-7402) co-cultured with normal human liver cells (HL-7702) and retinal vascular endothelial cells (RF/6A) [43]From human diploid cells (MRC-5), HL-7702 cells, and RF/6A cells cultured for 28 daysTranswell assay, western blotting, immunofluorescence, colony formation, qRT-PCR
   -
Bel-7402 co-cultured with HL-7702 or RF/6A ↓ migration and invasion of Bel-7402, ↑ epithelial marker (E-cadherin–catenin complex) expression, EMT-promoting transcription factors and mesenchymal markers.
   -
MRC-5-CM ↑ invasion and migration rate, inhibited tumorigenicity and viability and ↑ laminin and integrin expression on Bel-7402.
   -
The findings emphasized the powerful effects of TM on tumor growth via EMT and MET by influencing the expression of adhesion molecules such as laminins and integrins.
Primary REC [42]From nasal fibroblasts collected in 3D (3DCM) and 2D (2DCM) and cultured for 72 hAttachment assay, proliferation assay, cytoprotection, protein precipitation, SDS-PAGE
   -
3DCM ↑ cell attachment compared to 2DCM but not significantly.
   -
2DCM ↑ cell proliferation compared to 3DCM and control; however, the difference in all groups was not significant.
   -
2DCM and 3DCM ↑ cytoprotective effect.
   -
Protein concentration was higher in 3DCM compared to 2DCM.
   -
Conclusion: Cytoprotective effect by 3DCM on proliferating nasal fibroblast cells was equivalent to that of 2DCM.
Abbreviations: ↑ = increase; ↓ = decrease; FGF7 = fibroblast growth factor 7; COPD = chronic obstructive pulmonary disease; EMT = epithelial–mesenchymal transition; MET = mesenchymal–epithelial transition; IL-6 = interleukin 6; IL-8 = interleukin 8; MMP2 = matrix metallopeptidase 2; SPARC = osteonectin; CCR5 = C-C motif chemokine receptor 5; TNF-α = tumor necrosis factor α; VEGF = vascular endothelial growth factor; H2AXγ = phosphorylated H2A histone family member X; H₂O₂ = hydrogen peroxide.
Table
Table 2. Applications of CM from non-respiratory cells or tissues.
Table 2. Applications of CM from non-respiratory cells or tissues.
CELL TYPECM CHARACTERISTICSEXPERIMENTAL EVALUATIONRESULTS AND CONCLUSIONS
Human pulmonary mucoepidermoid carcinoma cells (NCI-H292) and MSC [57]From MSC stimulated with TNF-α and IL-1β (MSC-CM-STIM) and MSC only (MSC-CM) cultured for 24 hmRNA expression, ERK1/2 phosphorylation
   -
MSC stimulated with TNF-α and IL-1β ↑ FGF2, HGF, HBEGF, IL-6 gene expression.
   -
MSC-CM-STIM ↑ wound closure rate in NCI-H292.
   -
MSC-CM ↑ wound closure rate in ALI-PBEC culture compared to NCI-H292; MSC-CM-STIM exerted the same effect as DMEM-STIM in ALI-PBEC culture.
   -
MSC-CM-STIM ↑ ERK1/2 phosphorylation compared to control, which was mediated by the EGFR pathway.
   -
Inhibition of EGFR pathway ↓ MSC-CM-STIM effect on wound closure rate, proving EGFR was the predominant pathway.
   -
MSC are a viable option for cell-based therapy of inflammatory lung diseases such as COPD.
Lung carcinoma EC (A549), human bronchiole EC (BEAS-2B) [51]From adipose-derived stem cells (ADSC) cultured for 24 hImmunoblotting, fluorescence detection, cell migration assay
   -
ADSC-CM was protective against CSE and TGF-β1-induced cell death and EMT, as evidenced by ↑ cell viability and migration rates and ↓ LDH levels. This effect was associated with the maintenance of E-cadherin levels, which inhibited EMT activation.
   -
A549 in A549-CM and α-MEM had ↓ viability compared to A549 alone; CSE led to significant ↓ in cell numbers and ↑ LDH levels in A549.
   -
ADSC-CM prevented CSE-induced EMT and reversed the progressive decrease of epithelial marker expression associated with TGF-1 therapy. Stem cell-CM protected against cell death or CSE- and TGF-1-induced EMT, indicating its potential as a new target for lung cancer treatment.
Fetal distal lung EC (FDLE) [59]From umbilical cord mesenchymal stromal cells cultured for 72 hImmunofluorescence staining, cell permeability assay, chamber measurement, gene expression analyses, MTT
   -
MSC-CM ↑ Na+ transport, maximal ENaC, maximal Na+ and K-ATPase activity in FDLE monolayer.
   -
MSC-CM ↑ mRNA expression involved in Na+ transport, proving the earlier ↑ in Na+ transport.
   -
Sftpb and Sftpc ↑ in MSC-CM induced, aiding functional maturation.
   -
MSC-CM did not improve epithelial barrier function and proliferation.
   -
AKT and PI3K signaling contributed to MSC-CM-induced Na+ transport and ENaC activity.
   -
Rach1 signaling could be involved in MSC-CM-stimulated ENaC activity.
   -
MSC-CM significantly accelerated functional and structural maturation of fetal lungs, with effects mediated at least in part by the PI3K–AKT and Rac1 signaling pathways.
Lung carcinoma EC (A549) [52]From THP-1 monocyte-derived macrophages (mCM) cultured for 24 hELISA, western blotting, viability assay, immunocytochemistry
   -
PMA-treated mCM ↓ viability in A549 culture, triggered by inflammatory cytokines present in mCM, i.e., TNF-α and IL-8.
   -
Aesculetin ↑ viability in A549 + mCM compared to no aesculetin.
   -
Aesculetin ↑ E-cadherin in A549 with IL-8-induced mCM compared to no aesculetin, suppressing EMT induction on mCM.
   -
Aesculetin ↓ TIMP-1 and TIMP-2 expression in A549 with IL-8-induced mCM compared to no aesculetin.
   -
Aesculetin ↓ inflammatory macrophage infiltration in PHMG-induced mice compared to control.
   -
Aesculetin ↓ CXCR2 induction in PHMG-induced mice.
   -
Aesculetin exerted inhibitory effects on lung fibrosis and alveolar epithelial barrier breakdown caused by monocyte-derived macrophage infiltration.
Murine RAW 264.7 macrophage cells (RAW), lung carcinoma EC (A549), human breast cancer cells (MDA-MB-231), human colon carcinoma cells (COLO320HSR), human prostate cancer cells (PC3), murine lung carcinoma cells (344SQ) [62]From apoptotic murine lung carcinoma cell (ApoSQ) and monocyte-derived macrophages (MDM) cultured for 24 hImmunoprecipitation, western blot, migration assay, invasion assay, immunofluorescence, PPARγ activity assay
   -
RAW + ApoSQ-CM inhibited EMT on TGFβ1-induced 344SQ cells, demonstrating anti-EMT effect of ApoSQ when culturing RAW.
   -
ApoSQ-CM also inhibited EMT in A549, MDA-MB-231, COLO320HSR, and PC3.
   -
ApoSQ-CM inhibited migration and invasion of TGFβ1-induced 344SQ cells.
   -
MDM-CM exerted anti-EMT effect but did not inhibit TGF-β1-induced EMT marker changes, indicating the requirement for macrophage bioactive mediation.
   -
MDM-CM + ApoSQ prevented TGF-β1-induced cancer cell migration and invasion.
Lung carcinoma EC (A549) [58]From human placental MSC of fetal origin (hfPMSC) cultured for 48 hFlow cytometry assay, western blotting, Hoechst 33258 staining assay
   -
hfPMSC-CM exhibited antioxidant properties by ↑ total oxidant capacity while inhibiting O2− and OH, and protected A549 cells from apoptosis.
   -
hfPMSC can function as antioxidants in disease treatment.
Lung carcinoma EC (A549) [54]From 3T3-L1 (Ad-CM) co-cultured with A549 (Add-CM) for 48 hCell proliferation assay, colony formation assay, western blotting, wound closure assay, Transwell migration and invasion assays
   -
Ad-CM ↑ cancer cell migration and invasion, Add-CM ↑ A549 cell proliferation and colony formation compared to control and Ad-CM.
   -
The findings underlined the critical function of adipocytes in modulating lung adenocarcinoma A549 cell metabolism and suggested adipocyte participation in lung cancer development.
Lung carcinoma EC (A549) [55]From bone marrow-derived MSC (BMSC) cultured for 48 hTranswell migration assay, transmission electron microscopy, immunofluorescence analysis, western blotting
   -
BMSC-CM ↑ E-cadherin and CK8, ↓ vimentin and α-SMA compared to control.
   -
A549 + TGF-β1 ↑ vimentin and α-SMA, ↓ E-cadherin and CK8, ↑ p-Smad3, Snail1, COLI, and COLIII.
   -
BMSC-CM inhibited TGF-β1-induced EMT, demonstrating an antifibrotic effect on A549.
Lung carcinoma EC (A549) [60]From MSC cultured for 24 hImmunofluorescence, RT-PCR, proliferation, ELISA, wound repair assay
   -
MSC-CM inhibited fibroblast proliferation compared to control medium while ↑ epithelial wound closure compared to control medium.
   -
Finding shows the cells inhibited fibroblast growth while promoting epithelial healing; MSC amount was increased in fibrotic lung tissue compared to normal lung.
Lung carcinoma EC (A549) and small airway EC (SAEC) [56]From human umbilical cord Wharton’s jelly-derived MSC (hUC-MSC) cultured for 72 hProliferation assay, wound healing assay, Transwell, migration assay, western blotting, mass spectrometry.
   -
MSC-CM ↑ A549 proliferation and ↑ SAEC proliferation.
   -
MSC-CM ↑ wound healing compared to control group.
   -
MSC stimulated EC healing by releasing a repertoire of paracrine factors via JNK and P38 MAPK activation.
Lung carcinoma EC (A549) [53]From human leukemia monocytic cells (THP-1) cultured for 24 hWestern blot, ELISA, EMSA, luciferase assay
   -
Wogonin inhibited A549 cells induced by THP-1 CM in migration assay.
   -
THP-1 CM ↑ EMT in A549 via IL-6 expression.
   -
Wogonin neutralized EMT gene expression changes from THP-1 CM.
   -
Wogonin inhibited tumor cell migration in an inflammatory microenvironment, which could be potential treatment against tumor metastasis.
Human nasal EC (HNEC) [64]From S. aureus (S. aureus-CM) cultured for 48 hTEER, electron microscopy, immunofluorescence
   -
S. aureus CM affected the airway epithelium by disrupting the TJ between primary HNECs grown at an ALI.
Lung carcinoma EC (A549) [61]From BMSC cultured for 24 hFlow cytometry, cell proliferation assay
   -
MSC-CM + A549 ↑ proliferation and ↓ apoptosis compared to control.
   -
MSC protected against lung damage and fibrosis.
Lung carcinoma EC (A549) [65]From lipopolysaccharide (LPS)-treated A549 cultured for 12 hLactate dehydrogenase determination, apoptosis assessment, biomarker implication
   -
A549-CM ↑ apoptosis at 15% and 25%, ↑ necrosis at 25% compared to control.
   -
A549-CM ↑ IL-6, MIP-2, and MMP-9 level.
   -
LPS-treated human lung EC generated inflammatory mediators capable of inducing a translational clinically meaningful and damaging response in brain cells.
apoLung carcinoma EC (A549) [63]From UAP-BA- or ROFA-treated cells cultured for 24 hSpectrophotometric assays, superoxide anion generation, cytokine production
   -
Exposure to ROFA-CM and UAP-BA-CM inhibited A549\viability and affected their cellular response. ROFA-CM ↓ superoxide anion production; UAP-BA-CM ↑ O2 radical and IL-8 production in A549.
   -
The findings supported the concept that particle-induced lung inflammation and illness may include mediators produced from the lung.
Lung carcinoma EC (A549) [36]From human MSC (h-MSC) cultured for 24 hWound repair assay, MTT assay, mass spectrometry, migration assay
   -
MSC-CM ↑ wound repair in SAEC, ↑ migration in both SAEC and AEC, and ↑ AEC proliferation when supplemented with 10% FBS.
   -
hMSCs and/or their secretory proteins could be used to treat idiopathic pulmonary fibrosis (IPF) and other fibrotic lung illnesses.
Abbreviations: ↑ = increase; ↓ = decrease; LDH = lactose dehydrogenase; ENaC = epithelium sodium channel; PHMG = polyhexamethylene guanidine; CXCR2 = chemokine receptor 2; DMEM = Dulbecco’s modified Eagle’s medium; TNF-α = tumor necrosis factor alpha; IL-1β = interleukin-1 beta; EGFR = epidermal growth factor receptor; TGF-β1 = transforming growth factor beta 1; Na & K-ATPase = sodium potassium pump; Sftpb = surfactant protein-B; Sftpc = surfactant protein C; AKT = protein kinase B; PI3K = phosphoinositide 3-kinases; Rac1 = Ras-related C3 botulinum toxin substrate 1.
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MDPI and ACS Style

Muhammad Firdaus, F.I.; Nashihah, A.K.; Mohd Fauzi, M.B.; Manira, M.; Aminuddin, S.; Lokanathan, Y. Application of Conditioned Medium for In Vitro Modeling and Repair of Respiratory Tissue. Appl. Sci. 2023, 13, 5862. https://doi.org/10.3390/app13105862

AMA Style

Muhammad Firdaus FI, Nashihah AK, Mohd Fauzi MB, Manira M, Aminuddin S, Lokanathan Y. Application of Conditioned Medium for In Vitro Modeling and Repair of Respiratory Tissue. Applied Sciences. 2023; 13(10):5862. https://doi.org/10.3390/app13105862

Chicago/Turabian Style

Muhammad Firdaus, Fairuz Izan, Ab. Karim Nashihah, Mh. Busra Mohd Fauzi, Maarof Manira, Saim Aminuddin, and Yogeswaran Lokanathan. 2023. "Application of Conditioned Medium for In Vitro Modeling and Repair of Respiratory Tissue" Applied Sciences 13, no. 10: 5862. https://doi.org/10.3390/app13105862

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