INTRODUCTION

Tissue engineering is a multidisciplinary field that applies engineering principles to the biological sciences with the aim of developing substitutes for native tissues to restore, maintain, or improve their function (1). Nowadays, it represents one of the most promising areas of regenerative medicine, as artificial tissues constitute a realistic alternative for the treatment of a wide range of diseases, helping to overcome challenges such as donor shortage and immune rejection (2).

Nevertheless, achieving widespread clinical application of tissue engineering requires overcoming significant limitations, including the complexity and length of biofabrication protocols, as well as the lack of immediate availability of the constructs (3). This need is particularly pronounced in clinical contexts characterized by treatment urgency or difficulties in obtaining viable biopsies.

Oral mucosal lesions represent a significant example of such clinical scenarios. These lesions may arise from infectious, traumatic, congenital, or oncological conditions and compromise the structural and functional integrity of the oral cavity (4). Conventional treatments are based on surgical interventions using homologous autografts, heterologous autografts of skin or gastric mucosa, or allogeneic transplants; however, all these approaches present relevant limitations, including the limited availability of oral mucosa, the appearance of undesirable secretions or appendages, and donor scarcity, respectively (4–6).

In response to these limitations, various tissue-engineered oral mucosa models have been reported, ranging from keratinocyte monolayers to more complex constructs composed of keratinocytes and fibroblasts embedded within a scaffold (5). These models have demonstrated promising experimental outcomes and significant potential for clinical application (7). However, there is a growing interest in the immediate availability of prefabricated constructs that are ready for use while maintaining high structural and functional quality.

In this context, cryopreservation emerges as a viable strategy to ensure large-scale production and long-term storage of artificial tissues. This process involves the use of very low temperatures to preserve living cells and tissues in a structurally intact state (8). For cryopreservation to be effective, the use of cryoprotective agents is essential (8). These molecules reduce the biochemical and structural damage associated with freezing by modulating water transport, ice nucleation, and ice crystal growth (8).

Although the use of cryoprotective solutions is well characterized and optimized for cell suspensions, tissue cryopreservation remains a significant challenge due to the coexistence of multiple cell types, differences in permeability, and the properties of the extracellular matrix (10). These challenges are even more pronounced in tissue-engineered constructs, as the cryopreservation process is further influenced by the properties of the biomaterials used for their fabrication (7).

Regarding the cryopreservation of artificial oral mucosa, previous studies have evaluated the viability of cryopreserved oral mucosa stromal constructs, reporting promising results in terms of appropriate rheological parameters (7). However, the effects of cryopreservation on stromal cell viability and metabolic activity have not yet been thoroughly characterized. To date, the literature does not define a specific protocol for the cryopreservation of this substitute that ensures the preservation of fibroblast morphology and functionality. Therefore, further analysis and optimization of long-term cryopreservation methods are required as a critical step toward the clinical development and implementation of tissue-engineered oral mucosa substitutes.

The aim of this study is to investigate the effects of different cryopreservation protocols on the histochemical characterization of the stroma of tissue-engineered human oral mucosa. Specifically, diverse parameters were analyzed after freezing and reconditioning in order to evaluate four fundamental aspects that determine cellular functionality: metabolic activity, proliferation, extracellular matrix remodeling, and apoptosis.

MATERIALS AND METHODS

1. Cell culture preparation for the generation of artificial oral mucosa stroma.

Human oral mucosa fibroblasts (HOMF) were isolated from small oral mucosa biopsies obtained from healthy donors. The biopsies were subjected to enzymatic digestion of the stromal tissue using a 2 mg/mL type I collagenase solution at 37 °C for 6 hours under agitation. The resulting cell suspension was subsequently centrifuged and cultured. Primary HOMF cultures were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics and antimycotics (DMEMc).

2. Generation of oral mucosa stromal substitutes.

Stromal substitutes were obtained by combining human plasma with DMEMc containing a total of 2.25·10⁶ fibroblasts, resulting in a final density of 5·10⁴ cells per gel, and tranexamic acid. Subsequently, solutions of 1% CaCl₂ and 2% agarose were added to the mixture. Final volume was seeded into 24-well plates to obtain gels with approximately 1 mL of preparation per well. After allowing the gels to polymerize, they were covered with a small volume of DMEMc.

The stromal substitutes were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ for 21 days, during which the culture medium was changed every 3 days.

3. Preparation of cryoprotective solutions.

Cryoprotective agents were diluted to prepare the different experimental groups, and the solutions included were 10% DMSO, 10% Glycerol, 0.35 M Trehalose and CryoStor®. Also, PBS, a non-cryoprotective agent, was used as a negative control (CTR -). Once prepared, the solutions were sterilized by filtration.

4. Cryopreservation of oral mucosa stromal substitutes.

After completion of the culture period, different experimental groups were established according to the cryoprotectant used (10% DMSO, 10% Glycerol, 0.35 M Trehalose and CryoStor®), storage temperatures (4, – 20 and – 80 ºC), and the duration of cryopreservation (7, 15 and 30 days). Non-cryopreserved stromal substitutes were used as a positive control (CTR +).

The gels were immersed in the previously prepared cryoprotective solutions. Freezing was performed using a controlled-rate cooling method based on gradual temperature reduction by means of isopropyl alcohol–filled freezing containers.

5. Thawing and reconditioning of stromal substitutes.

For thawing, cryopreserved gels were brought to room temperature, the cryoprotectant was then decanted, and DMEMc was added to wash the samples and remove residual cryoprotectant. The gels were subsequently transferred to a culture plate with DMEMc and subjected to a reconditioning process for 48 hours at 37 °C in a humidified atmosphere containing 5% CO₂.

6. Sample processing.

Samples were fixed in 3.7% (w/v) formaldehyde, paraffin-embedded and sectioned at 5 µm thickness.

7. WST-1 assay.

Cell metabolic activity was evaluated using the WST-1 assay. 10% (v/v) WST-1 reagent was added to culture medium obtained after the reconditioning period, according to the manufacturer’s instructions. Samples were incubated for 4 h at 37 °C in a humidified atmosphere with 5% CO₂. Subsequently, 100 µL of the supernatant from each well was transferred to a 96-well plate for spectrophotometric analysis. Formazan dye absorbance, which correlates with the number of metabolically active cells, was measured using a scanning microplate reader at 450 nm, with 690 nm as the reference wavelength.

8. Immunohistochemical evaluation.

Immunohistochemical assays were performed to evaluate Ki-67 and MMP-14 quantification. The assays were conducted following standard immunohistochemical staining protocols and according to the conditions recommended by each manufacturer, revealed using diaminobenzidine and contrasted with hematoxylin.

9. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL).

To assess and compare apoptosis in the cells, a TUNEL assay was performed following the protocol provided by the manufacturer of the commercial kit (DeadEnd™ Fluorometric TUNEL System, Promega).

10. Image analysis.

The images obtained were analyzed by ImageJ image processing software, using the IHC Profiler plugin and the Split Channels tool.

RESULTS

The application of the described protocols allowed the successful generation, cryopreservation, and recovery of oral mucosa stromal substitutes. The results are presented according to metabolic activity, cell proliferation, extracellular matrix remodeling, and apoptotic activity.

1. Metabolic activity assessment.

Metabolic activity was evaluated at 7, 15, and 30 days of cryopreservation (Figure 1).

Figure 1. Evaluation of metabolic activity of human oral mucosa stromal substitutes under different cryopreservation conditions. Colored bars represent the absorbance at 450 nm per construct and the horizontal line indicates the absorbance observed in the non-cryopreserved control (CTR +).

At 7 days, constructs preserved at 4 °C showed metabolic activity comparable to non-cryopreserved controls, whereas at −20 and −80 °C, glycerol- and trehalose-treated constructs exhibited a marked reduction. In contrast, DMSO and CryoStor^® maintained higher metabolic activity at freezing temperatures. Constructs cryopreserved in PBS (CTR−) showed severely reduced activity under all conditions. After 15 and 30 days, metabolic activity progressively decreased in glycerol- and trehalose-preserved constructs, while DMSO and CryoStor^® consistently preserved metabolic function at −20 and −80 °C. CryoStor^® showed the best overall performance at long-term freezing conditions.

2. Cell proliferation assessment.

Cell proliferation was assessed by quantification of Ki-67–positive cells and total fibroblast count (Figure 2). Conditions showing a minimal number of recovered cells were considered non-representative and were excluded from comparative analysis.

Figure 2. Immunohistochemical evaluation of human oral mucosa stromal substitutes under different cryopreservation conditions for Ki-67 protein. Scale bar: 20 µm. Human foreskin tissue was used as a technical control to confirm staining efficacy. Colored bars represent the total number of cells per construct, while black dots indicate the percentage of Ki-67–positive cells.

After 7 days of cryopreservation, stromal substitutes preserved with trehalose at −20 and −80 °C, glycerol at −20 °C, and the PBS control at −80 °C showed a marked loss of fibroblasts. At 4 °C, glycerol-treated constructs exhibited the highest cell numbers, whereas trehalose-preserved constructs showed Ki-67 positivity comparable to non-cryopreserved controls. At −20 and −80 °C, CryoStor^® consistently yielded higher cell numbers and increased proportions of Ki-67–positive cells, reaching values equal to or exceeding those observed in non-cryopreserved constructs. Similar trends were observed after 15 and 30 days of cryopreservation, with CryoStor^® showing the most stable preservation of proliferative capacity, particularly at −80 °C.

3. Extracellular matrix remodeling assessment.

MMP-14 expression was quantified as a percentage of positive tissue area (Figure 3). Non-cryopreserved constructs showed a baseline positivity of 17%.

Figure 3. Immunohistochemical evaluation of human oral mucosa stromal substitutes under different cryopreservation conditions for MMP-14 protein. Scale bar: 20 µm. Human uterine tissue was used as a technical control to confirm staining efficacy. Colored bars represent the percentage of MMP-14–positive area per construct and the horizontal line indicates de percentage of MMP-14 positivity observed in the non-cryopreserved control (CTR +).

After 7 days of cryopreservation, constructs preserved with trehalose at 4 °C and glycerol at −80 °C displayed MMP-14 levels comparable to controls, whereas CryoStor^® at 4 °C showed markedly higher values. At longer cryopreservation times (15 and 30 days), MMP-14 positivity generally decreased across conditions, although specific combinations, such as glycerol at 4 °C and DMSO at −20 °C, showed increased values relative to controls.

4. Apoptotic activity assessment.

Apoptotic activity was evaluated by TUNEL assay in samples preserved under freezing conditions only (Figure 4).

Figure 4. Evaluation of apoptotic activity in cryopreserved human oral mucosa stromal substitutes by TUNEL assay. TUNEL-positive nuclei (green fluorescence) indicate apoptotic cells, while total nuclei are counterstained with DAPI (blue). Scale bar: 20 µm. Colored bars represent the total number of cells per construct, while black dots indicate the percentage of TUNEL–positive cells.

After 7 days of cryopreservation, constructs preserved with DMSO and CryoStor^® showed lower percentages of TUNEL-positive cells compared to glycerol and trehalose, particularly at −20 °C. At 15 and 30 days, glycerol and trehalose-preserved constructs exhibited a high proportion of apoptotic cells at all temperatures, whereas CryoStor^® maintained lower apoptotic levels, especially at −80 °C.

DISCUSSION

Cryopreservation is proposed as a key strategy to overcome the limitations related to the immediate availability of tissue-engineered constructs, with the aim of facilitating their clinical translation (8). However, the application of these methods requires strict control of both the storage temperature and the cryoprotective agents used, in order to avoid compromising cellular structural integrity and functionality. An efficient cryopreservation process is defined as one that preserves cellular metabolic activity, enabling cell proliferation and the ability to synthesize and remodel the extracellular matrix, while preventing the induction of apoptosis (7). Based on this premise, the results obtained after the application of the described protocols are discussed below for each of these functional aspects.

First, this study evaluated cellular metabolic activity as an indicator of viability potential. After 7 days of cryopreservation at 4 °C, HOMS constructs showed metabolic activity comparable to that of non-cryopreserved controls. However, prolonged storage at this temperature resulted in a progressive decline in metabolic activity, indicating a time-dependent loss of cellular functionality. These findings highlight the strong influence of cryopreservation temperature on cellular metabolism, particularly under non-freezing conditions. At freezing temperatures (−20 and −80 °C), constructs cryopreserved with glycerol and trehalose exhibited metabolic activity comparable to the negative control. This effect may be explained by the limited membrane permeability to glycerol and the extracellular mode of action of trehalose, which reduces their ability to protect stromal fibroblasts from cryo-induced damage. In the case of trehalose, its inability to penetrate cells may increase susceptibility to intracellular ice formation and thermal stress, especially during prolonged cryopreservation (11). Nevertheless, after short-term cryopreservation at −80 °C, trehalose-treated constructs showed partially preserved metabolic activity. This observation may be attributed to the formation of a vitrified extracellular matrix at ultra-low temperatures, which can reduce ice crystal formation and mechanical stress during freezing and thawing (12). Such effects may contribute to transient preservation of mitochondrial function during short-term storage, although they appear insufficient for long-term cryopreservation.

Then, cellular proliferation was analyzed, as it is an essential factor for achieving optimal regeneration of native tissue. The results demonstrate that cell proliferation is significantly affected by temperature, as observed in the constructs cryopreserved in PBS, in which the number of fibroblasts decreased as temperature decreased. In both the 7 and 15-day protocols, better outcomes were observed at refrigeration temperature (4 °C) when trehalose and glycerol were used as cryoprotective agents. This may be attributed to the fact that trehalose is a non-toxic cryoprotectant that does not induce adverse effects on cells at room temperature, unlike other cryoprotective agents (13). In the case of glycerol, its effectiveness may be explained by its action both inside and outside the cell; however, its limited ability to penetrate biological membranes reduces its protective effect at freezing temperatures (7,14). In contrast, at lower temperatures (−20 and −80 °C), DMSO and CryoStor^® showed superior results in terms of cell proliferation, with CryoStor^® yielding proportions of proliferative cells two- to three-fold higher than those obtained with DMSO. Several studies report that this commercial cryoprotectant is designed to maintain cellular ionic balance under hypothermic and freezing conditions, enabling rapid cellular recovery while reducing stress and cryopreservation-induced damage (15). Although DMSO is a highly effective cryoprotective agent due to its strong cell membrane permeability, its benefits are mainly observed under ultra-low temperature conditions, as its high cytotoxicity in the liquid phase limits its application at higher temperatures (16,17). Furthermore, when long-term cryopreservation (up to one month) was performed, a clear trend of increased cell proliferation with decreasing temperature was observed when CryoStor^® was used. Specifically, at −80 °C, DMSO, trehalose, and CryoStor^® showed high cell numbers and percentages of proliferative cells comparable to those observed in non-cryopreserved constructs.

Another crucial aspect of tissue regeneration is the ability of cells to remodel the extracellular matrix, a fundamental cellular function required to maintain tissue structure and function while preventing fibrosis (18). The results demonstrate that this capacity appears to be significantly reduced when no cryoprotective agent is used. Overall, the best results, most similar to the non-cryopreserved construct, were obtained under refrigeration conditions using trehalose, and at low temperatures when DMSO and CryoStor^® were used, specifically at −20 °C, as well as glycerol at −80 °C. A significant reduction in MMP-14 protein expression was observed in the experimental constructs compared with the non-cryopreserved sample in the two longest cryopreservation protocols (15 and 30 days). This finding indicates a deterioration in the cells’ ability to remodel the matrix over time, which may be explained by cryopreservation-induced physiological changes that slow down synthetic activity (19). Consequently, stromal substitutes subjected to prolonged cryopreservation protocols may require longer reconditioning periods.

Finally, evaluation of apoptotic activity in the cells composing the stromal substitutes is a key factor in confirming the viability of the artificial tissue as an advanced therapy medicinal product for clinical use. For this reason, a sensitive method for apoptosis detection, the TUNEL assay, was employed. In this case, only protocols performed under freezing conditions were evaluated. For short-term cryopreservation (7 days), all constructs exhibiting an acceptable total number of cells also showed high apoptotic activity, with the best results obtained using CryoStor^® and DMSO as cryoprotective agents, the latter being particularly effective at −20 °C. These findings are consistent with those observed for cell proliferation, suggesting that these two solutions may be optimal cryoprotective agents for the cryopreservation of tissue-engineered oral mucosa stromal substitutes. Previous studies have demonstrated that both agents result in reduced ice crystal formation within the construct microenvironment, thereby improving cellular preservation (20). Regarding the data obtained after 15 and 30 days of cryopreservation, the results indicate that in constructs preserved with glycerol or trehalose that maintained cellularity, all cells were undergoing apoptosis, whereas this percentage was markedly lower in constructs cryopreserved with DMSO and CryoStor^®. This relationship highlights the lower membrane permeability of glycerol and trehalose compared with DMSO-containing cryoprotective solutions. Once again, consistent with the previously discussed results, the use of CryoStor^® at −80 °C stands out positively, showing minimal apoptotic activity in the preserved cells. These observations are in agreement with the results obtained in the other assays.

In summary, the results presented in this study highlight the complexity of standardizing a cryopreservation protocol for tissue-engineered artificial tissues. In general terms, dimethyl sulfoxide–based solutions, such as DMSO and CryoStor^®, consistently demonstrated superior preservation of cell proliferation and extracellular matrix remodeling functions, while maintaining cell viability and preventing apoptosis. However, these outcomes are clearly dependent on both temperature and duration of cryopreservation. Therefore, future research should continue to address this issue through the use of more sensitive techniques that provide easily quantifiable results, thereby contributing stronger evidence toward the establishment of robust and well-defined cryopreservation protocols.

CONCLUSIONS

This work demonstrates the feasibility of cryopreserving tissue-engineered constructs such as human oral mucosa stromal substitutes, provided that appropriate cryoprotective agents and storage conditions are applied. The results indicate that dimethyl sulfoxide–based cryoprotectants, including DMSO and CryoStor^®, are the most effective options, as they preserve cellular proliferative capacity, extracellular matrix remodeling ability, and metabolic activity, while inducing minimal apoptosis when used under freezing conditions. Among them, CryoStor^® consistently showed superior performance, particularly in minimizing apoptosis-induced cell death and maintaining cellular functionality during long-term cryopreservation. Trehalose was identified as a potentially viable alternative under refrigeration conditions or short-term cryopreservation at ultra-low temperatures, where partial preservation of metabolic activity was observed. Glycerol did not exhibit a consistent protective effect across the evaluated temperatures and storage times, showing limited capacity to preserve cellular metabolism and functionality. Overall, these findings support cryopreservation as a key strategy to enable large-scale manufacturing, long-term storage, and immediate availability of tissue-engineered oral mucosa substitutes for clinical application. Nevertheless, further research is required to optimize and standardize cryopreservation protocols, particularly with respect to balancing cryoprotectant composition, temperature, and storage duration, to ensure the long-term preservation of both cellular viability and functional performance.

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ARTICLE INFORMATION

Conflict of Interest: The authors of this article declare that they have no conflicts of interest of any kind regarding the content of this work.

Correspondencia: Silvia Aguilar Pérez. Department of Histology, University of Granada. Avenida de la Investigación, 11 · 18016 Granada, Spain. E-mail: silviaaguilar@correo.ugr.es