Method standardization of secretome production, collection, and characterization: New insights and challenges

Abstract

Stem cell transplantation remains at the centre of regenerative therapy. Recent, studies have demonstrated that the therapeutic effect of stem cells originates from paracrine reactions from cellular products (secretome) rather than direct cell differentiation. The secretome (cell-free therapy) has the potential to eliminate the various challenges stem cell therapy encounters including apoptotic, inflammation, and non-immunogenic effects. Cell-free therapy is a new emerging field with the number of clinical trials slowly increasing, however, vast research currently is required in this field regarding standardization of protocols used to obtain the secretome. The recent progression of research in secretome production and characterization has created new perceptions of cellular communication and protein dynamics. Standard methods of secretome production, reproducibility, optimization, and bulk production remain a major challenge for scientists. Precise identification of biomarkers and therapeutic targets, through bioinformatics, mass spectrometry, and cell-based systems are promising techniques addressing these limitations. Therefore, this review aims to report on the methods for secretome production, collection, and characterization. Breakthroughs in this field of research will open the doors for personalized medicine, regenerative therapies, and other biotechnological applications.

1. Introduction

The incidence and prevalence of individuals living with degenerative disorders (spinal cord injury, traumatic brain injury, stroke, Parkinson’s disease, coronary artery disease, and cancer) is ever increasing [1]. These disorders are extremely challenging to treat due to their inherently low regeneration capacity [2]. In addition, these disorders activate other biological pathways that include cellular dysfunction, death, overactivation of immune cells, production of reactive oxygen species, and glial scar formation which aggravates serious outcomes compared to the primary event [3]. Current treatment and intervention approach for degenerative disorders include a combination of drugs and palliative care, which only alleviates the progression of the disease and does not reverse or intervene with the degenerative process [1]. Novel therapies such as cellular transplantation are being explored, however, despite their promising results, multiple challenges still need to be resolved which include transplantation safety, efficiency, and even determining their mechanism of action [4]. Multiple studies that have explored stem cell transplantation for spinal cord injury, Parkinson’s disease, and cancer models have reported therapeutic effects from the secreted cellular products rather than the differentiation of cells [[5], [6], [7]].

The cellular products referred hereafter as secretome [8], include proteins, growth factors, cytokines, chemokines, enzymes, and exosomes which contain RNA, lipids, and proteins. [9]. The study of the secretome and its influence on cell communication, disease, progression, and therapeutic efficacy is referred to as secretomics. The first secretomics studies originated from bacteria and fungi [10]. Studies report that their secretome composition was dependent on various pathologies and environmental factors [1]. Recently researchers have extensively studied the secretome of mammalian cells for therapeutic and diagnostic applications as the cellular products target the regeneration of damaged cells. [11]. These regeneration events are governed by exosomes from the secretome that enable cell-to-cell communication events that include paracrine signalling, hormonal communications, extracellular matrix interactions, immune responses, cellular repair regeneration, and waste management [12]. Examples of these communication events include the production of growth factors that can stimulate cell proliferation in neighbouring cells, the release of hormones that allow cell-to-cell communication over longer distances and across different tissues, migration, and differentiation, cytokines and chemokines released by immune cells elicit immune responses that modulate inflammation, cellular regeneration, and repair factors signal surrounding cells to activate the repair process, and enzymes produced in the secretome aid in removing damaged proteins and dead cell debris to maintain a healthy microenvironment for existing healthy cells [13].

The field of secretome therapy is new and emerging. The advantages of using secretome therapeutics include tissue protection, anti-apoptotic, anti-inflammatory, anti-scarring, immunomodulation, angiogenesis, and anti-tumour effects [4]. The most common sources of secretomes include umbilical cord tissue, bone marrow, peripheral blood, adipose tissue, and placental tissue [8]. Despite the therapeutic effect and advantages of secretome therapy, limitations in clinical translation exist which include standardization of the secretome formulation, characterization of bioactive compounds present, dosing, optimization of delivery, and mode of action [14]. To date there has been limited if no studies reported on secretome production from neuronal cells [15]. Secretome production is an inexpensive approach as it can be isolated and purified from cell cultures (cell free therapy) as compared to stem cell transplantation [10]. Therefore, evaluating current techniques and development of standardized lab scale production will enable proper scale up in bioreactors, thus, reducing overall costs of stem cell transplantation [13]. This review, therefore, will assess the current methods of production, collection, and characterization of the secretome from mesenchymal cells to ensure standardization and formulation of safe, efficacious, reproducible, and functional neurotherapeutics.

2. Secretome production

Multiple methods of secretome harvesting have been reported, however, the methods and conditions used for the formulation are not standardized [16]. This is critical as it serves as the foundation of secretome efficacy. The effects of multiple parameters can be evaluated to manipulate the type and quantity of molecules present in the secretome [3]. These include two-dimensional (2D) and three-dimensional (3D) cell culture (spheroid formation/organoid), and environmental factors (oxygen concentration, biochemical, drug stimuli) [4]. It is always important to note that culture conditions such as the physiological properties of the cell will increase both the yield and therapeutic function of the secretome gene expression [17,18]. To date, the effects of biochemical compounds, and drug stimulation on secretome production have not been completely analyzed and fully understood.

2.1. 2D and 3D cell culture

Although, the standard platform for cell expansion is a 2D cell culture [19,20], 3D cell culture methods used to produce secretomes is rapidly gaining popularity as it most closely mimics the physiological environment of cells [21]. This method allows cells to interact with residing cells in a 3D manner. There are multiple 3D culture systems which include spheroids, synthetic scaffolds, and extracellular matrixes [18]. It has been reported that spheroids limit oxygen entry to the inner cells thus, creating a hypoxic environment thereby, enhancing anti-inflammatory, anti-angiogenic, and tissue regeneration properties [20]. One of the most interesting approaches in secretome production is the encapsulation of cells in hydrogels for secretome production [3,4,16,18,20] (Fig. 1). Hydrogels offer immediate tuning of the cell’s microenvironment in terms of molecular properties, elasticity, and stiffness which directly influence the enhancement of secretome production [22].

Fig. 1.

Overview of secretome harvesting collection, processing, and storage using 2D and 3D harvesting methods [4,14,23,34].

A study by Otto and co-workers, 2023 [23], compared the impact of 2D and 3D cell formats on secretome production and their efficacy in the mineralization of a natural collagen scaffold. The results obtained showed that secretomes produced using both 2D and 3D microtissue models resulted in the accumulation of collagen, however, the secretome produced from the 3D microtissue model displayed enhanced mineralization with a homogenous distribution across the scaffold. Yang and co-workers, 2024 [24], used 3D hydrogels to produce secretomes and reported an increase in wound healing which may be possible due to an increase in the production of interleukin-10. These 3D models are easier to preserve, and their immediate readiness makes them excellent over-the-counter candidates [25].

2.2. Oxygen concentration

Standard cell culture techniques apply a normoxic oxygen concentration (21 % O₂) to cells, however, the physiological oxygen stress in cells ranges from 1 % (cartilage and bone) to 12 % in blood [26]. This is an indication that standard cell culture techniques employ oxygen concentrations higher than that of the cell’s physiological state [18]. According to the literature, to optimize secretome production and enhance its therapeutic efficacy, it is important to mimic the physiological state of cells [27]. Therefore, to achieve this goal studies have analyzed the secretome products under hypoxic O₂ concentrations (1–10 %) [28]. However, despite the differences in O₂ levels studies do not report on the effect on the promotion or demotion of extracellular vesicles [18]. Benefits of culturing in hypoxic conditions include maintenance of multipotency, enhancement of proliferation, regenerative, or cytoprotective effects [26]. Cells can respond to hypoxic environments by the upregulation of hypoxia-inducible factor 1-α (HIF-1α) [22]. This factor binds to the promoter regions of genes responsive to hypoxic environments, increasing the levels of glucose thus allowing longer survival periods [29]. Neovascularization is one of the most crucial factors in the regenerative process of damaged tissues [27]. Therefore, the enhanced therapeutic efficacy of cells produced in hypoxic conditions may be due to the production of vascularization factors induced by HIF-1α (vascular endothelial growth factor, angiotensin) [30].

2.3. Biochemical stimuli

The addition of biological factors (growth factors and cytokines) into the growth medium stimulates interactions between biomolecules and their receptors, thus increasing the production of proteins, miRNA, lipids, and metabolites. Previous studies have reported stimulation of cells with inflammatory factors (interferon-γ and tumor necrosis factor-α) that increased anti-inflammatory and regenerative factors that include prostaglandin E, interleukin-6, transforming growth factor, vascular endothelial growth factor, hepatocyte growth factor, and granulocyte colony-stimulating factor. As a result of increased anti-inflammatory factors, the cell’s metabolism is redirected to the glycolytic pathway thus sustaining the secretion of immunomodulatory factors and inhibiting proliferation of natural killer cells and CD4⁺/CD8⁺. A study by Lee and co-workers, 2015 [31], observed the promotion of M2 macrophage activation and tissue regeneration when the secretome of enhanced anti-inflammatory factors was administered in-vivo. Another study observed an increase in production and minor changes to the structure of extracellular vesicles when stimulated by interferon-γ and tumour necrosis factor-α [27]. However, this review focuses on whole secretome production and therefore, the following leading articles can be accessed for more information on extracellular vesicles [22,27].

Hydrogen peroxide may also be used to induce stress which in turn enhances expression of proangiogenic proteins [vascular endothelial growth factor and hepatocyte growth factor). A study by Bai and co-workers, 2018 [32], used an in-vitro model to determine the effect of stromal-derived factors on mesenchymal cells. The study revealed protection of mesenchymal cells from apoptosis enhanced cell proliferation, migration, and survival rates, thus indicating that biochemical stimuli enhance mesenchymal cell production, however, further studies are required to assess how the dosage and duration of exposure affect the secretome profile [18].

3. Secretome collection, processing and storage

The protocol for formulating secretomes includes harvesting stem cells without foetal bovine serum (FBS) to minimize interferences from proteins supplemented with FBS. The principal protocol for harvesting of secretomes includes the following steps [14]. Initially, (i) cells are grown in the presence of FBS to 60–80 % confluency, (ii) the cells are then washed several times with phosphate buffer saline (PBS), and (iii) serum-free medium is then added to the cells and incubated for a certain period before secretome harvesting and processing [4,23]. Fig. 1 below displays a detailed schematic representation of this process using both 2D and 3D cell culture. One of the most important factors during secretome harvesting is the incubation period before harvesting as this directly influences the secretome profile [33]. This factor varies across studies and therefore, it is of utmost importance that the incubation period before secretome harvesting is optimized for different cell types. Usually, cells are only able to tolerate starvation for a short period (12–48 h), however, starvation conditions are different for each cell type, and therefore, require optimization [34].

The medium change from serum rich medium to conditioned medium (serum free medium) is an intricate step. The cells are rinsed thoroughly to ensure all serum content is eliminated. A study by Pellitteri-Hahn and co-workers, 2006 [35], used rat endothelial cells to evaluate three techniques of changing from 20 % FBS medium to conditioned medium. Of the three methods, the most effective technique was two rinses with Dulbecco’s phosphate buffered saline (DPBS), and one rinse with serum free medium. Regardless of the multiple washing steps to remove serum proteins before secretome harvesting, a minimal amount of serum proteins remains in the culture making it challenging to obtain highly purified secretome. Studies report that the number of times cells are split has a direct effect on secretome composition, however, protein analysis of neuronal cell secretomes split (S3, S6, S9, S12) showed no significant difference [36].

Proteins present in secretomes are usually at very low concentrations, therefore, secretomes require further processing to concentrate the medium and prevent noise during analysis [37]. A simple cost-effective method which does not require specialized equipment and can effectively precipitate proteins is precipitation with trichloroacetic acid or acetone, however, disadvantages includes the loss of protein due to incomplete solubilization, inability to precipitate proteins present at low titres, and co-precipitation of contaminants. Furthermore, most proteins in secretome are in their soluble form; therefore, to retain solubility one should refrain from methods that involve protein precipitation [35]. Alternative methods include centrifugation using molecular weight cut off filters, dialysis, and evaporation using a freeze dryer; however, the drawbacks of the first two methods include protein binding to the membrane filter resulting in loss of protein due to the hydrophobic regions and functional groups of proteins that interact with the surface of the membrane. The third method, evaporation after centrifugation, is the most suitable method to concentrate the secretome as this eliminates loss of protein and protein precipitation. It is also important to note that although protocols are optimized for each cell line it is highly recommendable to determine cell viability and the percentage of dead cells in a conditioned medium to ensure that intracellular proteins released by necrotic and apoptotic cells do not distort the quantification of secreted proteins [38].

Both previous and more recent studies report similar protocols for secretome production. A study by Teixeira and co-workers, 2013 [39], harvested mesenchymal cells in a medium supplemented with FBS until 80–90 % confluence was obtained, the cells were then washed thoroughly in PBS and incubated for another 24–48 h in serum-free medium supplemented with glutamine. After incubation the cells were collected centrifuged and stored at −80 °C. Muntiu and co-workers, 2023 [41], produced a secretome from human amniotic cells. The cells were initially grown for 5 days in 0.5 ml DMEM supplemented with 2 mM l-glutamine. After incubation the cells were centrifuged at 300 g, filtered through a 0.22 μm filter, and lyophilized. More recent studies by Rogulska and co-workers, 2024 [40], and Perera and co-workers, 2022 [5], replaced 50 % of the conditioned medium with fresh medium after 24 h during secretome production to ensure the concentration of growth factors and analytes.

As per routine, clinical settings preserve samples at −80 °C in liquid nitrogen before analysis to ensure preservation with high-quality [42]. Secretomes on the other hand can be lyophilized by freeze drying which provides benefits such as secretome concentration, reduced volumes allowing more space for storage, and the possibility of storage at higher temperatures for longer periods [43]. Going ahead, the use of lyophilized concentrated secretomes in clinical settings will allow for larger clinical trials which will also accommodate facilities that lack specialized cell culture equipment and −80 °C liquid nitrogen storage [44]. A study by Rogulska and co-workers, 2024, studied the effect of storage duration (3 and 30 months) and temperature (−80 °C, −20 °C, and 4 °C) by monitoring the growth factors and cytokines in a lyophilized mesenchymal stromal secretome. The study showed that biomolecules were preserved at both 3 and 30 months at −80 °C. At −20 °C three growth factors were reduced, and at 4 °C and room temperature a further reduction of five growth factors was observed. However, the lyophilized secretome supplemented with trehalose improved the reduction of growth factors at both 4 °C and room temperature. There is currently a lack of standardization in secretome storage conditions as current lyophilized mesenchymal cell secretomes are stored at all three of the above-mentioned temperatures until use [45], however, the study by Rogulska and co-workers, 2024 [40], confirms that maximum preservation of growth factors for both long and short periods can only be achieved at −80 °C storage.

4. Analytical methods used to characterize secretomes

There are multiple techniques both quantitative and qualitative (Fig. 2) used to characterize cell secretomes which include serial analysis of gene expression (SAGE), DNA microarrays, antibody and bead-based methods, mass spectrometry, RNA sequencing, and bacterial/mammalian secretion traps (Table 1) [12].

Fig. 2.

Diagram summarizing methods used in quantitative and qualitative secretome analysis.

Table 1.

Advantages and disadvantages of analytical methods used to characterize secretomes.

Method	Advantage	Disadvantage
SAGE	•Direct and qualitative technique used to measure gene expression •Detects both known and unknown transcripts •No hybridization is required	•Time consuming •Identification can be difficult as the same tag may be present on multiple genes •mRNA levels do not represent protein levels •Difficult to detect low quantity transcripts •Identification is limited to the number of tags sequenced •Errors in sequencing
DNA microarray	•Quantitative •High throughput •Cheaper than RNA sequencing	•Does not yield complete coverage •Requires prior knowledge of the sequences •Only detects proteins expressed at the mRNA level. •Difficult to identify rare transcripts. •Gene expression is not always correlated with protein expression •Smaller dynamic range
Protein microarray	•High sensitivity •Broad reproducibility	•Expensive •Antibodies may bind non-specifically to proteins •Rapid denaturation
Mass spectrometry (gel-based methods)	•Identification of unknown proteins •High resolution	•Limited reproducibility •Limited dynamic range •Low throughput •Inability to detect proteins at low quantity proteins
Mass spectrometry (non-gel-based methods)	•Detection of low titer proteins •Improved sequence coverage •High throughput •Reproducible	•ICAT only detects proteins with cysteine •SILAC cannot be used in clinical samples •Difficult to detect cancer biomarkers with SELDI-TOF-MS
RNA sequencing	•Requires low RNA quantities •Low background noise •No prior sequence knowledge required •Non-model organisms can be used	•Only sequence information can be obtained •Bioinformatics analysis difficult •Rare transcripts are underrepresented •Difficult to detect cancer markers using SELDI-TOF MS •Artifacts are introduced at different stages of data processing
Signal sequence traps	•Detection of all secreted proteins	•Time consuming •Intensive labor required •Traps are not widely used in mammalian cell secretome studies

4.1. Serial analysis of gene expression (SAGE)

The method of SAGE entails RNA isolation and cDNA synthesis with oligo DT primers. Thereafter, cDNA is digested with enzymes and bound to streptavidin beads [17]. The beads are then ligated, cleaved with a tagging enzyme creating blunt ends, and amplified using specific primers [46]. The tags are then cloned and sequenced using high throughput sequencing [35]. This method measures the global gene expression patterns and identifies genes by 9–10 bp tags [47]. Like all methods, the advantage of this method is that it is direct and qualitative for measuring gene expression, able to detect both known and unknown transcripts, and does not require hybridization [48]. The disadvantage is that it is time consuming, identification of genes is difficult as the same tag may be present on multiple genes, mRNA levels do not represent protein levels, limited by the total number of tags that can be sequenced, sequencing errors, and low quantity transcripts may be difficult to identify [49].

4.2. DNA microarrays

There are two types of DNA microarrays genechips, and spotted arrays. The GeneChip method consists of approximately 25 bp oligosaccharides synthesized directly onto the chip at high density [14]. Hybridization is detected using a one colour fluorescent light for the detection of gene expression [50], spotted arrays use a two-colour system to measure gene expression [1]. The cDNA is synthesized and spotted on glass slides and labelled Cy3 and Cy5. RNA molecules are hybridized to the array resulting in a two-colour fluorescence [51]. The DNA microarray technique is high throughput, quantitative, and less expensive compared to RNA sequencing, however, does not yield complete coverage, requires prior knowledge of the sequences, may only detect proteins that are expressed at the mRNA level, gene expression does not always correlate with protein expression, smaller dynamic range, and identification of rare transcripts are difficult [41].

4.3. Protein microarrays

In this technique, proteins are immobilized onto slides and captured using label based or sandwich molecules [52]. Some commercial kits may contain known secretory proteins, which send out fluorescent or chemiluminescence signals [36,53]. A fluorescent multiplexed format is then used to detect secretory proteins. DNA and protein microarrays are highly sensitive and have a broad reproducibility [17]. On the downside they are expensive, antibodies may bind non-specifically to proteins, and rapid denaturation [54].

4.4. Mass spectrometry

In this method, proteins are first separated by liquid chromatography and thereafter, detected by mass spectrometry [55]. Gel dependent methods allow for the identification of unknown secretory proteins and display high resolving power [56]. This method utilizes 2D and differential electrophoresis followed by mass spectrometry [57]. The most versatile detection method includes SDS-PAGE followed by LC-MS [58]. This has been used to detect secretory proteins in both plants and mammalian cells. In 2D electrophoresis a discrete spot is obtained by separating the protein by its net charge and then dimension, thereafter the discrete spot is subjected to mass spectrometry [59]. Differential electrophoresis measures differential protein expression by labeling proteins with fluorescent dyes (Cy2, Cy3, and Cy5) after which the gel runs in two dimensions. Protein is detected by a superimposed fluorescence image [57]. Unfortunately, this method also displays drawbacks that include limited reproducibility, limited dynamic range, low throughput, and inability to detect proteins at low levels [60].

There are four gel independent methods namely, isotope-coded affinity tag (ICAT), isobaric tag for relative and absolute quantization (iTRAQ), stable isotope labelling by amino acids in cell culture (SILAC), and SELDI-TOF MS [14]. These gel independent methods allow for the detection of proteins at low titers, through isotopic protein labelling which improves sequence coverage, are high throughput, and reproducible [49]. In the ICAT method, ICAT reagent is used to analyze two protein samples [36]. The reagent binds to cysteine residues, which have a heavy and light isotope, and a biotin tag. The samples are then mixed, digested, and recovered by the biotin tag through affinity chromatography, thereafter, LC-MS is conducted [33]. iTRAQ uses four tag reagents that bind to the N-terminus and can detect the relative abundance of proteins in up to four samples in one mass spectrometry experiment. This method has been used to detect the secretome of the influenza A virus [61]. Identification of plant and mammal secretomes depends on the SILAC methods which labels two samples that lack amino acids with light and heavy isotopes [17]. The samples are then combined, trypsin digested, and subjected to LC-MS. SELDI-TOF MS has been used to detect the secretome of stromal cells this is a high throughput method that directly separates proteins in the SELDI protein chip which is then subjected to LC-MS [14]. All the methods mentioned above display uniqueness and can be effectively applied based on specific research requirements. Disadvantages are that ICAT only allows the detection of proteins with cysteine, SILAC cannot be used in clinical samples, and it is difficult to detect cancer biomarkers using SELDI-TOF MS.

4.5. RNA sequencing

RNA sequencing can be used for multiple analyses including transcriptome analysis, unravelling the secretome profiles of an organism, and monitoring gene expression changes during development, or under different conditions. In this method, mRNA is used to synthesize cDNA, which is fragmented and sequenced using various sequencing platforms [62]. After sequencing reads are aligned to a reference genome and a transcriptome map is generated. The advantage of this method is that low RNA quantities are required, low background noise, no prior knowledge of the gene is required, and even non-model organisms can be used [63]. However, only sequence information can be obtained from this method, bioinformatic analysis is challenging, most reads represent common RNA which means identification of most common secretory proteins, rare transcripts are underrepresented, and artifacts are introduced during different stages of data processing [64].

4.6. Secretion or signal sequence traps

Mammalian and bacterial secretion traps are not used in most laboratories as this method is time consuming and laborious [17]. In the mammalian secretion trap the cDNA is fused to a mammalian expression vector which directs the secretion of TAC fusion protein which is monitored by anti-CD25 antibody. Cells are transinfected with plasmids and the secreted fusion proteins are detected by immunostaining [65]. The reporter gene used in bacterial traps include c-myc or β-lactamase, however, these traps have not been widely used in mammalian secretome studies [65].

5. Conclusion and future perspectives

Secretome therapy is much faster and bypasses the immunocompatibility issues experienced with stem cell therapy. Although this method is safe for clinical usage there is a large gap in research that needs to be completed regarding the optimization method for secretome production, purification, and characterization. Although multiple methods have been used to characterize the secretome majority of the secretory proteins are present in very low concentrations resulting in isolation and identification difficulties. To overcome the above-mentioned challenges future studies should focus on identifying biomolecules and understanding the organization of their biological systems using spatial omics (proteomics, genomics, and metabolomics) which may result in the elucidation of more accurate disease mechanisms and therapeutic targets eliminating off target effects. Drug delivery systems using biopolymers should also be considered for the transport of the secretome which may offer greater stability, prevent off target deliveries, allow controlled release rates, enhance solubility, and improve safety and therapeutic efficacy of the secretome.

Informed consent statement

Not applicable.

Author contributions

All authors contributed to the conceptualisation and design of this review. The literature search and first draft of the manuscript were performed and written by NS. PK and YEC provided methodology and scientific and technical assessments as well as editorial input. All authors read and approved the final manuscript.

Declaration of competing interest

The authors declare no conflict of interest.