Epithelial regenerative activity of topic administration of canine adipose-derived mesenchymal stem cells secretome in eye drops format—an in vitro study

Introduction

Background

The mesenchymal stem cells can originate from a lot of tissues, such as bone marrow, dental pulp and adipose tissue. The latter is particularly favored in view of the abundance and easy extraction for cellular isolation, originating the adipose-derived mesenchymal stem cells (ADSCs) (1). These cells have the ability to undergo differentiation into a range of cellular lineages, according to the metabolic requirement and exogenous stimuli (2). In veterinary medicine, there are a lot of fields of research and clinical administration of them, due to the commercial viability and ethical permission related to the isolation, cultivation, and administration of these cells (3). Independent of the target-tissue administration, the cells’ potential is related to the local signalizing through the secretion of signaling compounds and tissue-stimulating molecules, which are related to the extracellular vesicles (4,5).

The secretion products, named secretomes, are formed by proteins and cytokines, among other components, which are responsible for cellular signaling and desirable tissue effect (6). According to this pathway of action from ADSCs, the purification and directly administration of secretome may be a pharmacological alternative to the stem cells therapies, resulting in enhanced desirable outcomes and reduced potential side effects, as well as facilitating manipulation, storage, and application of the final product (6,7). Related to the power of action in different cellular lineages and tissues, there are countless targets of treatment using the mesenchymal stem cells’ products (6). Moreover, given the significant caseload of ophthalmic diseases (8), the production of new pharmacological therapies for the eye is an important research target and commercial opportunity.

The corneal injuries are very common in the canine ophthalmic routine (9), including inflammatory, infectious, and degenerative diseases and, principally, the corneal ulcers, that is mainly caused by trauma, being necessary the multifactorial therapies for therapeutic success (10-12). Related to the poor vascular formation of the corneal tissue and delayed tissue repair, the use of different products aiming to upgrade the epithelial tissue repair is necessary and, sometimes, obligatory (13,14). In this field, the ADSCs and their secretome may figure as an important modality in upgrading the corneal therapies, in view of positive effects in the tissue-repair previously proved for another superficial tissues (7).

Rationale and knowledge gap

However, there is no previous scientific evidence proving the power of canine ADSC’s secretome, as eye drops, to the repair of cornea’s epithelial histologic structure. The authors hypothesize that if the secretome derived from canine cells shows favorable outcomes in treating human injuries in vitro, it could offer a novel and promising approach for alternative therapies in dogs. Furthermore, its effectiveness could serve as a predictive model for the potential utilization of human ADSC’s secretome in similar allogeneic treatments.

Objective

This way, the aim of this study was to evaluate and to characterize the tissue effect of canine ADSCs’ secretome in human epithelial cells in vitro, manipulated as eye drops and administered as therapeutic approach for human HaCaT cellular lineage, as concept proof. We present this article in accordance with the MDAR reporting checklist (available at https://amj.amegroups.com/article/view/10.21037/amj-23-214/rc).

Methods

Cellular culture and secretome production

The adipose-derived stem cells, for secretome production, were obtained from a commercial batch (BIO CELL Cellular Therapies Ltd., Brasilia, Brazil). The cells, extracted from adipose tissue of healthy dogs, were acquired through elective surgical procedures for sterilization. The total of 2.5×10³ cells were added to DMEM culture medium, in culture flasks of 25 cm² at 37 ℃ and 5% CO₂, changing the medium every 48 hours, until the confluence of 70%. After the desirable confluence, the cells were trypsinized and expanded to culture bottles of 75 cm². For secretome acquisition, adapted from previous studies (15,16), the culture of the cells was done at hyperflask bottles of 1.720 cm², summing 1.4×10⁷ cells per bottle with bilateral adhesion to DMEM medium, with 10% of fetal bovine serum (FBS), at controlled atmosphere 5% CO₂ and 37 ℃, changing the medium every 48 hours, until the confluence of 90%. So, when the 90% confluence was achieved, the cellular washing was done with DMEM-phosphate-buffered saline (PBS) and the culture medium was changed to modified-DMEM without phenol red and serum-free (without FBS) every 48 hours.

After that, the supernatant was collected and divided in conic tubes of 50 mL for centrifugation at 3,000 g during 30 min to remove cells and cellular debris. Sequentially, a new centrifugation at 17,000 g was taken, for 40 min, to remove apoptotic structures. The supernatant was collected again and filtered in a mesh of 0.22 µm, to remove major structures. So, the supernatant was transferred to ultrafiltration devices, with a mesh of 100 kDA, where was one more time centrifuged at 4,000 g for 5 min to remove the water excess, and the final product was a concentrated secretome, that was used for the eye drops production. The entire process was done for three different cellular batches, originated from three different donators, and the final product of the three processes were homogenized, resulting in secretome pool, which was combined with other compounds to get three eye drops, to triplicate the assays.

ADSCs secretome’s eye drops production

The eye drops used in this research was composed by basic excipient associated to the secretome pool produced. Three eye drops batches, named C1, C2 and C3, were produced in order to triplicate the results, and the unique difference between them was the moment of production. The excipient was composed by sodium hyaluronate 15 mg/mL associated with sodium citrate 10 mg/mL, citric acid 10 mg/mL and sorbitol 45 mg/mL, with a final volume of 10 mL per eyedrop, summing the excipient and the secretome solution.

Laboratorial culture of human keratinocytes cellular lineage (HaCaT)

The cells used for the in vitro proofs were HaCaT human keratinocytes (RRID:CVCL_0038), acquired from a commercial cell bank (BCRJ 0341) and cultured in laboratorial environment, in culture bottles of 75 cm³. For the cultivation, it was used the DMEM medium high-glucose, added to 10% of FBS. The bottles of cells were inserted at an incubator with 5% CO₂ and 37 ℃. The culture medium was changed every 48 h. When 80% confluence was acquired, the trypsinization was done using TrypLE™ Express (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and the cells were transferred to the assays.

Cellular metabolic activity assay (MTT proof)

The metabolic activity was verified through the MTT metabolization ability of the cells (17). The assay was prepared using 5 mg of MTT (Invitrogen, Ref. M6494, Carlsbad, CA, USA), solved in 1 mL of sterile DMEM-FBS, filtered with syringe and a mesh of 0.22 µm and storage in −20 ℃. For MTT proof, a total of 100 µL of DMEM medium high-glucose, with a 1×10⁴ HaCaT cellular concentration per well, was added to 96 well-plates (Corning 3599, Glendale, AZ, USA). The well-plates were placed at an incubator with 5% CO₂ and 37 ℃ per 24 h to cellular adhesion. After adhesion period, the cells received the eye drops treatment (C1, C2 and C3) in a proportion of 10% of the total volume inserted in each well and compared with the control group without treatment (18).

After 24 h of the treatment, the medium was removed from the wells, and 100 µL of DMEM high-glucose medium with MTT (5 mg/mL) in 10% concentration was added in each well in the plate. The plates were stored at incubators per 4 h to guarantee MTT metabolization and formation of formazan crystals. After 4 h, the culture medium was exchanged to 100 µL of dimethyl sulfoxide (DMSO) for crystals dissolution. The ELISA equipment (DR 200BS model, Kasuaki, Wuxi, China) was used for the 570 nm absorbance analysis. The cellular viability was defined using the ELISA absorbance results, with white-control normalizing, by using mathematical formulas in Excel software.

Scratch-wound assay

For the scratch-wound assay, 12 wells (Corning 3516) were adopted in order to repeat three times the experiment, which ones were filled out with 5×10⁴ HaCaT cells per well and DMEM culture medium. The well-plates were storage at incubators 5% CO₂ and 37 ℃ per 24 h for cellular adhesion and monolayer formation. After 24 h, the plates were removed and analyzed using inverted optical microscopy for cellular monolayer confirmation. Sequentially, the bottom of plates was marked with permanent marker to define limits for photography registration at standard locations.

The assay began with the creation of an artificial scratch wound in the cellular monolayer, followed by the analysis of cellular migration towards the center of the wound to ensure its repair, as previously conceptualized (19). The results of control and treated (C1, C2 and C3) groups were compared. The scratches were created using sterile pipette tips (P1000), positioned at 90°, in a single-linear scratch for each well. After, the wells were washed with 1X phosphate-buffered saline (PBS) without FBS to remove the supernatant cells. Continuously, a permanent marker was used to demarcate the bottom plate’s boundaries for photograph acquisition at standard locations. The total of four markers were done, 90° with the wound and numbered sequentially to identify the point of photograph registration and guide the next one, as shown in Figure 1.

Figure 1 Permanent marker demonstration in the bottom of cultivation well-plate, done for the in vitro scratch-wound assay. 40× magnification. The position of the mark was used as superior limit for photograph acquisition of the different groups.

After washing, the cells received DMEM culture medium with 10% volume of eye drops (control and treated), for 24 h. The treatments were replicated in triplicate, with three wells designated for each type of eye drop (control, C1, C2 and C3). The photographs were acquired with inverted optical microscope, with 40× magnification, at T0 (before treatment) and T1 (after 24 h of the treatment). All photograph acquisition was done after the washing and removing the supernatant, avoiding interferences, at standard position both times for migration evaluation. The images were inserted and analyzed with Image J Software (Software 1.481, Rayne Rasband, National Institute of Health, USA) using the “wound healing size tool” plugin. This approach enables the identification of the proportion of cellular migration towards the wound and monolayer repair, in comparison to the initial state (T0). To define the percentage of migration and wound closing, the software identified the changes in the closing area in relation to the lesion area (at T0), according to: Wound closing (%) = [A(0) − A(t)/A(0)]×100, being A(0) the area in T0, and A(t) the T1 area.

Cellular replication assay

For the replication assay, the HaCaT cells, at 7.5×10⁴ concentration per wells, were distributed in plates with 12 wells (Corning 3516), in order to triplicate the results for each group, with 1 mL of culture medium. The plates were storage at incubators 5% CO₂ and 37 ℃, per 24 h. After that, the culture medium was exchanged and the cells were treated with the eye drops (control, C1, C2 and C3) with 10% proportion of the culture medium volume per well.

It was performed three wells for each eye drop, and after 24 h of the treatment the cells were trypsinized with TrypLE (Gibco 12604-021), incubated for 20 min, inactivated with FBS supplemented medium and centrifuged at 1,300 rpm per 3 min. Sequentially, the cells were resuspended in 1 mL of culture medium and each sample was counted three times for each group, at Neubauer chamber, and automatic counter (Corning cell counter).

Statistical analysis

The data recorded in the assays (MTT, scratch-wound and replication) were submitted to unidirectional analysis of variance (ANOVA) test. The P values <0.05 were considered significant (95% confidence interval). The GraphPad PRISM 6 (GraphPad Software Ins., La Jolla, CA, USA) software was adopted for data analysis. The normal distribution was achieved with the Shapiro-Wilk proof. The independent results were presented as mean ± standard deviation.

Ethical consideration

A project license was deemed unnecessary by the ethics committee of the Catholic University of Brasilia, given the in vitro nature and commercial aspects of the cellular specimens used in this experiment.

Results

This study was conducted in vitro aiming to evaluate the regenerative epithelial potential of secretome’s eye drops to keratinocytes cellular lineages, for ophthalmic therapeutics. It adopted cellular metabolic proofs, wound repair assay and replication tests, and the results were presented in sequence.

Cellular metabolic activity assay (MTT proof)

The cellular metabolic assay was conducted for HaCaT cultures, for control and treated groups, with and without ADSC’s secretomes. The three secretomes eye drops (C1, C2 and C3) were tested separated. As results, there was no statistical difference (P>0.05) between C1, C2 and C3 groups. In addition, the treated groups showed upgrade in metabolic activity and cellular proliferation, with statistical significance (P<0.05), compared to the control group (Figure 2).

Figure 2 MTT proof for metabolic cellular assay to HaCaT cells of control and treated groups with ADSC’s secretome in eye drops format, after 24 h of the treatment. Comparison between the metabolic cell viability and the different groups. 95% confidence interval. There was no statistical difference between the secretomes eye drops (P>0.05, ns = not significant), however significant difference was noted between the control and treated group (P=0.02*). ADSC, adipose-derived mesenchymal stem cell.

Cellular replication assay

The HaCaT cells were evaluated according to the replication rates after 24 h of treatment, for control and treated groups. The treated C1 and C2 groups showed significant enhancing of the final cellular concentration compared to the control group. However, the C3 treated group showed a bigger final concentration of cells compared to the control group, but there was no statistical difference between them (Table 1).

Related to the dispending time necessary to the duplication of cellular counting among the groups, the control spent 24 h, while the treated groups needed less time, being C1 17 h, C2 16 h and C3 18 h.

Scratch-wound assay

After 24 hours of treatment with the eye drops on the cellular culture, a significant enhancement in the growth of the HaCaT monolayer was observed compared to the control group (Figure 3). This observation confirms the ability of the secretome eye drops to promote cellular migration and repair of epithelial tissue. Treatment with C1, C2, and C3 eye drops led to an average increase of 30% in epithelial cellular migration compared to the control cells. This enhancement aligns with the results observed in scratch-wound assays, which are indicative of improved wound repair in epithelial tissue (Figure 4).

Figure 3 Scratch-wound in HaCaT cells results for control and treated groups with three eye drops containing secretome from ADSC. Evaluation of cellular migration and epithelial tissue repair. Statistical analysis after 24 h of treatments, with 95% confidence interval. Statistical significant difference between control and treated group was identified, being C1 (P<0.001***) the main one, followed by C2 and C3 (P=0.001** and P=0.003**), respectively. ADSC, adipose-derived mesenchymal stem cell.

Figure 4 HaCaT monolayer evaluation during Scratch-wound assay, performed for the control (A,B) and (C-H) treated groups with ADSC secretome’s eye drops. 40× magnification. (A,C,E,G) The initial scratch-wound and demarcation of the cellular margin (C) (T0). (B,D,F,H) The migration of epithelial cells in direction to the wound after 24 h. ADSC, adipose-derived mesenchymal stem cell.

Discussion

Key findings

This paper aimed to evaluate the effect of eye drops containing ADSCs secretome to keratinocytes cellular lineage through in vitro proofs, hypothesized based on previous studies that proved the effectiveness of the compound in treating various tissues and injuries (20). It was possible to observe that the secretome eye drops was able to improve the cellular metabolism, cellular replication and same way, upgrade the migration rate in 30% to the iatrogenic wound of HaCaT cells. The power of signaling the target tissue, aiming to improve the cellular proliferation and the tissue repair in a lower time, is one ability of the secretome (2) and this information was confirmed through this research, getting support for the author’s hypothesis, that ADSC’s extracellular vesicles also may be effective for ophthalmic therapeutics. It was possible to demonstrate the efficacy of the secretome in repairing epithelial tissue, even when administered as topical medication eye drops. This finding may provide support for decision-making and guide future directions for additional researches and clinical applications of such eye drops in clinical practice and commercial use, owing to the promising results.

Strengths and limitations

As previous results showed (21), the epithelial effects of ADSC’s secretome were positive and promising for clinical use. Furthermore, when the corneal healing process is evaluated, it is known that the tissue is dependent of various signaling pathways, like the stimuli inherent to the stromal tissue (22), which was not able to be evaluated in this research due to the characteristics of an in vitro investigation. However, this kind of research are very desirable as pre-clinical studies to support further investigations. Added to this, new approaches to increase even more the wound healing process are emerging every time, resulting in new ways of therapies using ADSC’s secretome, and the treatment of mesenchymal stem cells before extracting secretome may figure as a new research and method for optimization of the healing time of complicated wounds (23). Knowing that, new research must be placed to verify the clinical usage of the compounds for its safety and reproducibility of the good results in vitro obtained, in order to investigate the synergism that the molecules may have with the local tissue signaling in vivo. As demonstrated by the results presented here, it is anticipated and hypothesized that the synergism between the endogenous and exogenous cells may figure as an approach to generate better results inherent to the tissue healing.

Explanations of findings and comparison with similar researches

Corneal tissue repair presents challenges due to its intricate mechanisms. The cellular migration is essential for this process, being more important than cellular replication, for the tissue regeneration (24). This fact supports the importance of the results presented here for the tested eye drops, which ones showed satisfactory effects of potentializing the cellular migration process and enhancing the wound repair. The exosomes, one of the compounds of the extracellular vesicles from ADSC, are one of the most popular research targets of mesenchymal stem cells, and figure as an important line of research for shallow and deep injuries in superficial tissues (24,25). When administered to corneal epithelium, it was able to potentialize the cellular proliferation and migration, besides promote inflammatory and local-tissue cell secretion modulation (26). On the other hand, these researches did not characterize the effect on the epithelium. The exosomes were also used associated to a topic chitosan gel for corneal injuries, revealing benefic power for cellular regeneration and migration at the epithelium, through miRNA’s secretion (27). Against the previous methodologies, this research aimed to use the entire extracellular vesicles, where the exosomes figure as a part of them, but are also associated with other factors, such as proteins, lipids and microvesicles. The secretome, with its myriad components, represents an important research target, given the various properties of its compounds. These properties may potentially lead to superior outcomes compared to the use of individual molecules alone, as demonstrated by the positive results observed in this study.

The secretomes derived from ADSC of dogs may be important to the reduction of corneal opacification after tissue repair, decreasing the extracellular matrix deposition during the repairing and increasing the wound cell filling through cellular migration (21), corroborating the importance of the results described in this paper for eye drops administration, that the influence of the products are favorable for avoiding future corneal opacifications, being one more benefic effect of topic use of ADSC’s secretome. Furthermore, the extracellular vesicles from corneal tissue, mainly secreted for endothelial cells, are positively related to the enhancement of epithelial cells migration and replication rates, principally keratinocytes, playing an important role in corneal tissue repair (27). Through this information, it is possible to hypothesize that the synergism between both extracellular vesicles, from endothelium and from ADSC’s secretome, may result in better results for the tissue regeneration and wound filling for cellular migration. This hypothesis was supported by previous studies showing synergism of the secretome from mesenchymal cells to the ones inherent to the local tissue, resulting in the desirable effect for damage repair (2). This way, the results may be encouraging for further investigations involving clinical trials and in vivo experimentation, aiming to investigate the possible better effects resulted from the local tissue signaling at the same time.

During the corneal injury process, it is expected that keratocytes undergo apoptosis, with subsequent secretion of signaling substances that will induce the cellular metabolism enhancement, in view of promoting tissue repair (28). Additionally, the complete epithelial regeneration of the wound can last for a very long time (29). Summing the facts of cellular metabolic enhancement’s necessity, for epithelial corneal injuries, and the results of improvement of metabolic rates of HaCaT, it is possible to prove the effectiveness of the secretome’s eye drops for an important pathway of tissue regeneration. The results indicated significant improvement in cellular metabolism for the treated groups across all three batches produced, as compared to the control group. It is known that the cytokines and interleukins secreted by the injured cells are the main substances involved in the paracrine action to other epithelial cells when a corneal injury took place (30). Allied to it, it is previously proved that the ADSC’s secretome have cytokines and interleukins as compounds (6), which may be one of the directly and indirectly related mechanisms to the enhancement of cellular metabolism through increasing the signaling of the local cells.

Besides the cellular metabolism upgrade, the cellular replication rates were also increased for the treated groups, resulting in lower time for the wound occlusion. The epithelial cellular replication is one of the necessary mechanisms to the wound filling of the corneal tissue, happening at the same time to cellular migration (28). The new-cells formation is impaired for the corneal vascular deficiency and, this way, has a secondary role in the regeneration process (24,28). In this context, the utilization of metabolic enhancers could be significant for expediting the process, as evidenced by the outcomes observed when HaCaT cells were treated with secretome eye drops. The improvement of cellular final counting for the C1 and C2 groups is a promising and positive result, showing the ability of the secretome to improve the cellular replication rates for epithelial tissue. Clinically, this effect plays an important role in accelerating the wound healing time.

Implications and actions needed

Through the evaluation of the in vitro potentials of secretomes derived from mesenchymal stem cells from dogs, it was possible to conclude that the alternative therapy is beneficial for corneal epithelial damages, when used like eye drops, due to its efficacy observed in stimulating HaCaT cells. The benefits were associated with reduced time required for wound closure, elevated cellular metabolism, and increased replication rates observed in the treated groups. These pathways are crucial for corneal epithelial healing. These results demonstrated the positive potential of the topic secretome therapies for the epithelial tissue, principally the cornea, encouraging the conduction of further future clinical trials.

Conclusions

The canine ADSC secretome’s eye drops were effective for the improvement of tissue growth, cellular migration and replication and metabolic activity enhancement for human keratinocytes, through in vitro assays. In vivo proofs, for the canine and human species, are encouraged for verifying the clinical safety and effectiveness of secretome’s eye drops for corneal healing.

Acknowledgments

Funding: None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://amj.amegroups.com/article/view/10.21037/amj-23-214/rc

Data Sharing Statement: Available at https://amj.amegroups.com/article/view/10.21037/amj-23-214/dss

Peer Review File: Available at https://amj.amegroups.com/article/view/10.21037/amj-23-214/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://amj.amegroups.com/article/view/10.21037/amj-23-214/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. A project license was deemed unnecessary by the ethics committee of Catholic University of Brasilia, given the in vitro nature and commercial aspects of the cellular specimens used in this experiment.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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