Metabolic Mode of Alginate-Encapsulated Human Mesenchymal Stromal Cells as a Background for Storage at Ambient Temperature

Abstract

Introduction:

Human mesenchymal stromal cells (MSCs) are attractive for both medical practice and biomedical research. Nonfreezing short-term storage may provide safe and simple transportation and promote the practical use of MSCs.

Objectives:

We aimed to determine the duration of efficient storage at ambient temperature (22°C) of human dermal MSCs in different three-dimensional organization and to investigate the role of cell metabolic mode in the resistance to the ambient storage damaging factors.

Methods:

MSCs in monolayer, suspension, and encapsulated in alginate microspheres (AMS) were stored in sealed containers at 22°С in culture medium. Viability (fluorescein diacetate /ethidium bromide) and metabolic activity (Alamar Blue assay) were assessed at 0, 3, 7, 10, and 14 days of the storage. Mitochondrial membrane potential (JC-1 test), cell cycle analysis, reactive oxygen species level, and resistance to hydrogen peroxide were analyzed under culture conditions.

Results:

Alginate encapsulation was shown to maintain viability (about 85%), metabolic activity, and adhesion ability during storage for 7 days. The storage of MSCs in both monolayer and suspension was less efficient. Culture of MSCs in AMS decreased basal metabolic activity, mitochondrial activity, and led to reversible cell cycle arrest compared to standard two-dimensional culture. MSCs in AMS have a lower basal level of reactive oxygen species and higher resistance to hydrogen peroxide compared with those in monolayer culture.

Conclusion:

Revealed shift into quiescent metabolic mode is essential for alginate-encapsulated MSCs resistance to storage at ambient temperature.

Introduction

Human mesenchymal stem/stromal cells (MSCs) are a promising cell type for clinical medicine and biomedical research due to their unique properties. MSCs can be isolated from a variety of tissue sources.^1,2 Besides the generally accepted osteogenic, adipogenic, and chondrogenic lineages,³ MSCs have been shown to differentiate into myocytes, cardiomyocytes,⁴ hepatocytes,^5,6 and neural cells.^7,8 High therapeutic potential of MSCs is supported by the secretome with angiogenic,^9,10 immunomodulatory,¹¹ and growth stimulatory action.¹² Practical use of MSCs requires effective preservation technologies. The only established approach for long-term storage is cryopreservation. However, it has limitations such as the need for expensive equipment and liquid nitrogen for storage and transportation, its effects on the cytoskeleton,¹³ the differentiation potential,¹⁴ and the gene expression rate.¹⁵ Various approaches to reduce dimethyl sulfoxide concentration have been investigated,^16,17 but it is still generally accepted that the negative effects of cryopreservation should be mitigated. The development of nonfreezing storage of MSCs is highly anticipated due to its simplicity and safety. It has been shown that some cell types may be stored under hypothermic conditions, but the data are contradictory. Thus, storage of MSCs in a monolayer at 4°C for 18 hours to 3 days resulted in increased apoptosis and necrosis.¹⁸ The study¹⁹ revealed that induced pluripotent stem cell-derived retinal pigment epithelium cells stored in suspension at 37°C exhibited the lowest viability due to hypoxia; those preserved at 4°C were damaged via microtubule fragility, while cells stored at 16°C were optimally preserved up to 6 hours. It was found²⁰ that the survival rate of MSCs in suspension stored for 6 hours at room temperature was lower compared to storage at 4°C. Conversely, the study²¹ demonstrated that MSCs stored in suspension for 6 hours at 4°C lost their viability. Several studies^22–24 have shown alginate encapsulation to be a promising approach for effective storage of MSCs at ambient temperatures. The mechanisms of alginate encapsulation protective effect are still not clear. Mahler and colleagues²⁵ hypothesized that alginate gel could provoke integrin ligation disrupting the integrin–caspase complex, thereby preventing integrin-mediated apoptosis. It is also widely accepted that alginate entrapment provides protection against mechanical stress.²⁶ However, the impact of encapsulation into alginate hydrogel on cellular metabolism remains poorly investigated.

We have chosen high-voltage electrospraying²⁷ for MSCs encapsulation because it operates in a controlled manner. Among the existing types, we focused on human dermal MSCs due to the notably less invasive tissue collection procedure compared to bone marrow and adipose tissue. Dermal MSCs possess similar multilineage differentiation potential and even higher growth and angiogenic capacities compared with bone marrow-derived MSCs.²⁸ Previously, we demonstrated that human dermal MSCs are positive for CD29, CD73, CD90, and CD105 and negative for CD34 and CD45,²⁹ and that alginate encapsulation supports their viability and differentiation potential.³⁰

Herein, we integrated our knowledge of MSC biology with expertise in cryotechnology to devise a freezing-free protocol for short-term MSCs storage. Additionally, we investigated the role of metabolic mode in cell resistance against storage-related damage. The design and idea of the study are shown in Figure 1.

FIG. 1.

The design and idea of the study.

Materials and Methods

Human skin-derived mesenchymal stromal cell isolation and culture

The MSCs were isolated by the explantation method,³¹ following the written informed consent of the donor in accordance with the Helsinki Declaration of the World Medical Association for Biomedical Research and the requirements of the IPC&C Ethics Committee. Cells were cultured in adhesive culture flasks (TPP, Switzerland) at 37°C, 5% CO₂, and 95% humidity in minimal essential medium-α modification (α-MEM, Biowest, France) with 10% fetal bovine serum (PAA, Austria), 50 μg/mL penicillin (Biowest, France), 50 μg/mL streptomycin (Biowest, France), and 0.2 mM l-glutamine (Sigma-Aldrich, USA). MSCs at passages 4–8 were used for experiments.

MSCs encapsulation in alginate microspheres

Alginate sodium (Sigma, 71238) 2% solution was mixed with a cell pellet (0.5 mL per 1 × 10⁶ cells). A high-voltage encapsulation device and a 0.4 mm diameter blunt cannula (100 Sterican®, B.Braun Melsungen, Germany) were used to disperse the mixture into a 100 mM solution of CaCl₂ via electrospaying. Alginate microspheres (AMS) were allowed to cross-link for 5 minutes, then stepwise washed out with Hank’s balanced salt solution (HBSS, Sigma). During the final washing-out step, the saline solution was replaced with a culture medium.

Preparation of samples with different MSC 3D-organization

To obtain a monolayer, cells were cultured for 5 days, detached by trypsinization, washed, seeded in 96-well plates at 12 × 10³ cells/well, and cultured overnight for adhesion. One plate was then analyzed, while the remaining plates were stored or cultured. For the suspension group, cells were cultured for 6 days, detached from the flask, samples were taken for analysis, and the remaining cells were stored.

To prepare AMS, cells were cultured for 5 days on a flask, detached from the flask, encapsulated, and cultured overnight in AMS for adaptation to encapsulated state, then samples were taken for analysis, and the remaining AMS were stored or cultured.

Storage at ambient temperature

MSCs in monolayer, suspension, and encapsulated in AMS were stored in sealed containers at 22°С in culture medium in a cooling thermostat LAUDA (LAUDA, Germany). Viability and functional state parameters were assessed before storage and at days 3, 7, 10, and 14 of storage (n = 3 in triplicate for each group).

Viability assessment

Viability was assessed by double staining with fluorescein diacetate (FDA)/ethidium bromide (EB).³² Fluorescent dye incorporation into cells was evaluated using LSM 510 META confocal laser scanning microscope (Carl Zeiss, Germany). Confocal images were obtained along the Z-axis with 20 μm intervals at an excitation wavelength of 488 nm for FDA and 543 nm for EB.

Metabolic activity assessment

The tested samples were incubated for 2 hours with culture medium containing 10% Alamar Blue (AB, Serotec Ltd., Bio-Rad, USA). The fluorescence of the reduced form of AB was measured using a Tecan GENios microplate reader (Tecan GENios, Austria) at an excitation wavelength of 550 nm and an emission wavelength of 590 nm and expressed in relative fluorescence units (RFU).

Attachment efficiency

The attachment efficiency was assessed in such a way: cell suspensions were seeded in 24-well plates (for the AMS group, cells were first extracted from alginate hydrogel using citrate solution), after 24 hours of culture the number of unattached cells was counted using a hemocytometer. Attachment efficiency was calculated using the formula: $e = \frac{m - n}{m} \times 100 %,$ where e is the attachment efficiency; m is the number of seeded cells; and n is the number of unattached cells.

Haematoxylin–Eosin staining

Samples were stained as described by Fischer et al.³³ and were examined using inverted microscope CETI EpiFluor (CETI, Belgium).

Mitochondrial activity (JC-1 test)

Samples were washed with HBSS and then incubated for 30 minutes with fluorescent cationic dye JC-1 at a final concentration of 2 μM. The fluorescence of J-aggregates was measured at an excitation wavelength of 550 nm and an emission wavelength of 590 nm, and the fluorescence of JC-1 monomer was measured at an excitation wavelength of 514 nm and an emission wavelength of 535 nm using a TECAN GENios microplate reader. The red/green fluorescence ratio was used as an index of mitochondrial activity.

Cell cycle analysis

MSCs in monolayer were cultured using Premo™ FUCCI Cell Cycle Sensor kit reagents for 24 hours. The cells were then collected, one half was seeded on tissue culture plastic, the rest was encapsulated in AMS. Live-cell imaging of cell cycle progression was performed using an Olympus FV10i-LIV confocal laser scanning microscope.

Reactive oxygen species level and resistance to hydrogen peroxide-induced oxidative stress

Reactive oxygen species (ROS) level was measured using Cellular ROS Assay Kit Deep Red (Abcam). Basal ROS level was assessed in MSCs cultured for 24 hours. MSCs cultured for 24 hours in monolayer or in AMS were exposed to 3 mM hydrogen peroxide solution for 2 hours to induce oxidative stress,³⁴ washed out with HBSS, and induced ROS level was determined. Fluorescence of ROS sensor was detected using confocal laser scanning microscope Olympus FV10i-LIV and Olympus cellSense Software.

Statistics

The obtained results were statistically processed. The normality of the data distribution was determined by the Shapiro–Wilk test. The significance of differences between groups was examined by Student t-test for independent variables. Data were represented as M ± m; differences were considered significant at p < 0.05.

Results

The viability of MSCs before storage was over 98% in all groups (Fig. 2a). In AMS, cell viability remained higher throughout the entire storage period compared to the other groups. Over the course of 7 days, viability exceeded 70%, meeting FDA requirements.³⁵ In monolayer storage, viability remained sufficiently high (above 70%) for 3 days, but significantly declined with further storage. The lowest viability was observed in suspension storage, dropping below 70% after 3 days of storage.

FIG. 2.

Viability (a) and metabolic activity (b) before (day 0) and during storage of MSCs in monolayer, suspension, and encapsulated in alginate microspheres at ambient temperature. *p < 0.05 in AMS compared to the data in suspension in corresponding storage day, #p < 0.05 in AMS compared to the monolayer in the corresponding storage day. AMS, alginate microspheres; MSCs, mesenchymal stem cells.

The metabolic activity of MSCs in AMS was 30% lower after 24 hours of culture before the storage compared to the other two groups (Fig. 2b, 0 day). However, during the storage, metabolic activity in the AMS group was supported in the most efficient manner. When stored in monolayer, metabolic activity of MSCs decreased by 50% by day 3 and continued to decline by 20%–25% every subsequent 3 days. The lowest metabolic activity was observed in cells stored in suspension, decreasing to nearly 15% of the initial level within the first 3 days.

To assess the impact of storage on the self-renewal potential of MSCs, we examined attachment efficiency and proliferation ability before and after 7 days of storage. Prior to storage, all groups exhibited high attachment efficiency (>99%). However, following 7 days of storage, a significant difference emerged. While MSCs stored in AMS maintained a high attachment efficiency (64.0 ± 3.6%) and displayed normal morphology, cells stored in suspension showed significantly reduced attachment (12.0 ± 0.8%) (Fig. 3b, c). Moreover, the lower attachment efficiency of suspension-stored MSCs correlated with impaired proliferation. Monolayer-stored MSCs were almost completely detached after 7 days (Fig. 3a), rendering them unsuitable for further analysis. These findings suggest that encapsulation in AMS efficiently preserves the self-renewal potential of MSCs after 7 days of storage.

FIG. 3.

MSCs after 7 days of storage at ambient temperature: detached cells in stored monolayer (a), cells returned to 2D culture conditions from stored suspension (b) and AMS (c), 24 hours of culture, hematoxylin–eosin. 2D, two-dimensional.

Thus, we have revealed that alginate encapsulation supported viability and self-renewal potential of MSCs stored at ambient temperature but decreased their basal metabolic activity in the first 24 hours before storage. Therefore, we decided to further investigate how alginate encapsulation and culture in AMS affect metabolic mode, cell cycle, and ROS metabolism as key factors in MSC resistance to storage-induced damage.

The mitochondrial activity of MSCs after culture in monolayer or in AMS for 3 days was assessed using JC-1 dye, which exhibits green fluorescence in its free form and red fluorescence in the form of J-aggregates.³⁶ It was revealed that the red/green JC-1 fluorescence ratio was 1.47 for MSCs in monolayer and 0.5 for MSCs cultured in AMS. Considering that a higher mitochondrial potential results in more J-aggregates, it can be concluded that alginate encapsulation decreased mitochondrial activity.

For cell cycle analysis MSCs were transduced with Premo™ FUCCI Cell Cycle Sensor constructs, encoding red (RFP) and green (GFP) fluorescent proteins fused to Cdt1 and geminin. In the G1 phase, only Cdt1-RFP is detectable, rendering cells red. Conversely, during the S, G2, and M phases, only geminin-GFP is expressed, resulting in green cells. The G1/S transition exhibits both Cdt1 and geminin, leading to yellow nuclei. Live imaging was utilized to visualize the cell cycle in MSCs growing in two-dimensional (2D) monolayer and in AMS 3D culture. Cells transduced with different phases of the cell cycle, predominantly in S, G2, and M phases (green cells), were either seeded on tissue culture plastic or encapsulated in AMS (Fig. 4, 1 hour of culture). During subsequent culture, MSCs were mainly in S, G2, and M phases in the monolayer, if sufficient space was available, while those encapsulated in AMS became arrested in the G1 phase 48 hours later (Fig. 4).

FIG. 4.

Live-cell imaging of cell cycle progression during culture of MSCs transduced with Premo™ FUCCI cell cycle sensor constructs in monolayer and AMS: green—S, G2, and M phases; red—G1 phase; and yellow—G1/S transition. (a) 1 hour of culture in monolayer. (b) 24 hours of culture in monolayer. (c) 48 hours of culture in monolayer. (d) 1 hour of culture in AMS. (e) 24 hours of culture in AMS. (f) 48 hours of culture in AMS.

Basal intracellular ROS levels, assessed as real-time ROS sensor fluorescence, were 32.8 ± 5.2 and 2.32 ± 0.25 RFU/cell in monolayer and in AMS cultured for 24 hours, respectively. Following a 2-hour exposure to 3 mM hydrogen peroxide, ROS sensor fluorescence increased to 154.5 ± 7.8 RFU/cell in monolayer culture and 11.78 ± 0.44 RFU/cell in AMS. Therefore, encapsulation reduced basal ROS levels and enhanced MSC resistance to oxidative stress.

Discussion

The optimal storage method for MSCs is still uncertain. Currently, cryopreservation is the gold standard, though the procedure and cryoprotective agents’ toxicity impact MSCs.³⁷ Nonfreezing storage is a promising alternative, but an optimal protocol is yet to be developed. Early studies showed hypothermic temperatures (2–10°C) were more favorable than room temperature for short periods (6–24 hours) for MSC suspensions.^38,39 Later, hypothermic and ambient temperatures were found to be similarly effective for storage.^40,41 To prolong viability for an extended period, two strategies affecting metabolism proved effective: MSC suspension storage in specific solutions^21,42,43 or storage in 3D constructs like alginate hydrogels.^{23,24,30,34,44,45} Given that “low” hypothermic temperatures suppress enzymatic systems responsible for antioxidant defense, genome and cell structure repair, and homeostasis, we opted for storage at ambient temperature. We then compared the effects of different 3D MSC organizations on storage efficacy. Our findings showed that encapsulation maintained high MSC viability for 7 days at 22°C, meeting the cell therapy standards of the US Food and Drug Administration. Encapsulation preserved adhesion, metabolic activity, and proliferation of MSCs stored for 7 days, whereas monolayer or suspension storage resulted in viability loss.

The next step was to identify mechanisms responsible for storage resistance. While previous studies^{23,24,30,34,44,45} also demonstrated the successful storage of MSCs in alginate hydrogels at ambient temperature, our study provides additional insights into underlying mechanisms of encapsulated cell resistance by highlighting the contribution of metabolic mode into enhanced storage capacity. We analyzed the changes in the metabolic pillars of cell functionality following encapsulation and subsequent culture. We observed a reduction in MSC metabolic activity 24 hours after encapsulation, a finding also reported by other researchers.⁴⁵ Using the JC-1 test, we detected decreased mitochondrial activity in MSCs cultured in AMS compared to monolayer culture. These findings align with results⁴⁶ showing higher glycolytic activity and lactate production in MSCs within nonadhesive alginate hydrogel compared to modified hydrogels that promote cell adhesion. The unmodified alginate used in our study limited interactions, resulting in round-shaped encapsulated cells with reduced metabolic and mitochondrial activities.

MSC proliferative behavior is influenced by 3D cell organization. We observed cell cycle arrest in the G1 phase 48 hours after encapsulation in AMS, corroborating findings⁴⁷ that culture in alginate hydrogel suppresses MSC proliferation. Low metabolic activity and reversible cell cycle arrest are key features of the natural quiescence state, which protects adult stem cells from damage and preserves stemness.^48,49 Similarly, resistance to nonfreezing storage may result from the quiescence induced by alginate encapsulation. Slight changes in alginate concentration and cross-linking conditions did not appear to affect the development of resistance to nonfreezing storage in encapsulated cells. Successful ambient storage of MSCs in alginate hydrogels has been demonstrated in both our study and others, using calcium-cross-linked hydrogels with alginate concentrations ranging from 1.2% to 2.2%^34,45 as well as strontium-cross-linked alginate hydrogel.²³

The damaging factors in hypothermic preservation include ion imbalance, pH shift, oxidative stress, and endoplasmic reticulum stress,^18,50 with oxidative stress becoming most critical under hypoxia. Evidence shows increased ROS generation during hypothermic storage and rewarming.^51–53 Given the decreased mitochondrial activity in encapsulated MSCs, we anticipated a lower basal ROS level, as the electron transport chain is the main ROS producer. Analysis using the intracellular ROS sensor deep red fluorescence confirmed a significantly reduced basal ROS level in encapsulated MSCs compared to monolayer. Lower basal ROS levels indicate a more balanced redox state, which can enhance the cell’s capacity to withstand oxidative stress. We actually found increased resistance to oxidative stress, with ROS levels after hydrogen peroxide exposure significantly lower in cells in AMS than in monolayer. Alginate polymers were shown to scavenge-free radicals⁵⁴ and suppress oxidative stress-induced apoptosis.^55,56 Mechanisms behind enhanced ROS resistance may be attributed to both the protective effects of alginate and metabolic adaptations induced by the microenvironment within the alginate matrix and need to be deeper delved in future studies. Our findings of changes in ROS metabolism in encapsulated MSCs have positive implications for enhanced cellular component protection and oxidative stress response during ambient storage.

Conclusion

In conclusion, we demonstrated the high survival rate of alginate-encapsulated human skin-derived MSCs during storage at 22°C in sealed containers for up to 7 days. Alginate encapsulation resulted in decreased basal metabolic activity, mitochondrial activity, and basal levels of ROS, leading to reversible cell cycle arrest. We also observed increased resistance to hydrogen peroxide-induced oxidative stress in encapsulated MSCs. Therefore, the process of alginate encapsulation initiates a series of metabolic changes that significantly contribute to cell resistance during hypothermic storage at ambient temperature and under hypoxic conditions.

Footnotes

Authors’ Contributions

O.P. and N.T. conceived of the study concept and design. N.T., O.H., Y.K., and O.T. carried out the experiments. Y.K., I.K., and K.K. participated in technical assistance. N.T., Y.K., and O.T. performed the statistical analysis. N.T., O.P., and O.T. took the lead in writing the article. All authors provided analysis of results, critical feedback, and helped shape the research and the article.

Author Disclosure Statement

The authors have no relevant affiliations or financial involvement with any organization with a financial interest in or financial conflict with the subject matter discussed in the article.

Funding Information

This study was supported by the National Research Foundation of Ukraine (project no. 2021.01/0276) and the National Academy of Sciences of Ukraine (project 2.2.6.145/no. 6 23.09.2021§ 24-5).

References

Baghaei

, Hashemi

, Tokhanbigli

, et al. Isolation, differentiation, and characterization of mesenchymal stem cells from human bone marrow. Gastroenterol Hepatol Bed Bench, 2017; 10(3):208–213.

Mushahary

, Spittler

, Kasper

, et al. Isolation, cultivation, and characterization of human mesenchymal stem cells. Cytometry A, 2018; 93(1):19–31; doi: 10.1002/cyto.a.23242

Dominici

, Le Blanc

, Mueller

, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement. Cytotherapy, 2006; 8(4):315–317; doi: 10.1080/14653240600855905

Young

, Steele

, Bray

, et al. Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors. Anat Rec, 2001; 264(1):51–62; doi: 10.1002/ar.1128

Weng

, Lin

, Hsiang

, et al. The effects of different growth factors on human bone marrow stromal cells differentiating into hepatocyte-like cells. Adv Exp Med Biol, 2003; 534:119–128; doi: 10.1007/978-1-4615-0063-6_9

Wang

, Wang

, Yan

, et al. Expression of hepatocyte-like phenotypes in bone marrow stromal cells after HGF induction. Biochem Biophys Res Commun, 2004; 320(3):712–716; doi: 10.1016/j.bbrc.2004.05.213

Sanchez-Ramos

, Song

, Cardozo-Pelaez

, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol, 2000; 164(2):247–256; doi: 10.1006/exnr.2000.7389

Yang

, Huang

, Ma

. Bone marrow stromal cells express neural phenotypes in vitro and migrate in brain after transplantation in vivo. Biomed Environ Sci, 2006; 19(5):329–335.

Estrada

, Li

, Sarojini

, et al. Secretome from mesenchymal stem cells induces angiogenesis via Cyr61. J Cell Physiol, 2009; 219(3):563–571; doi: 10.1002/jcp.21701

10.

Maacha

, Sidahmed

, Jacob

, et al. Paracrine Mechanisms of mesenchymal stromal cells in angiogenesis. Stem Cells Int, 2020; 2020:4356359; doi: 10.1155/2020/4356359

11.

Karaöz

, Demircan

PÇ

, Erman

, et al. Comparative analyses of immunosuppressive characteristics of bone-marrow, Wharton’s jelly, and adipose tissue-derived human mesenchymal stem cells. Turk J Haematol, 2017; 34(3):213–225; doi: 10.4274/tjh.2016.0171

12.

Harrell

, Fellabaum

, Jovicic

, et al. Molecular mechanisms responsible for therapeutic potential of mesenchymal stem cell-derived secretome. Cells, 2019; 8(5):467; doi: 10.3390/cells8050467

13.

Chinnadurai

, Garcia

, Sakurai

, et al. Actin cytoskeletal disruption following cryopreservation alters the biodistribution of human mesenchymal stromal cells in vivo. Stem Cell Reports, 2014; 3(1):60–72; doi: 10.1016/j.stemcr.2014.05.003

14.

Bahsoun

, Coopman

, Akam

. Quantitative assessment of the impact of cryopreservation on human bone marrow-derived mesenchymal stem cells: Up to 24 h post-thaw and beyond. Stem Cell Res Ther, 2020; 11(1):540; doi: 10.1186/s13287-020-02054-2

15.

Davies

, Smith

, Cooper

, et al. The effects of cryopreservation on cells isolated from adipose, bone marrow and dental pulp tissues. Cryobiology, 2014; 69(2):342–347; doi: 10.1016/j.cryobiol.2014.08.003

16.

Liu

, Xu

, Ma

, et al. Cryopreservation of human bone marrow-derived mesenchymal stem cells with reduced dimethylsulfoxide and well-defined freezing solutions. Biotechnol Prog, 2010; 26(6):1635–1643; doi: 10.1002/btpr.464

17.

Rogulska

, Tykhvynska

, Revenko

, et al. Novel Cryopreservation approach providing off-the-shelf availability of human multipotent mesenchymal stromal cells for clinical applications. Stem Cells Int, 2019; 2019:4150690; doi: 10.1155/2019/4150690

18.

Corwin

, Baust

, et al. Characterization and modulation of human mesenchymal stem cell stress pathway response following hypothermic storage. Cryobiology, 2014; 68(2):215–226; doi: 10.1016/j.cryobiol.2014.01.014

19.

Kitahata

, Tanaka

, Hori

, et al. Critical functionality effects from storage temperature on human induced pluripotent stem cell-derived retinal pigment epithelium cell suspensions. Sci Rep, 2019; 9(1):2891; doi: 10.1038/s41598-018-38065-6

20.

, Li

, Liao

, et al. Effects of storage culture media, temperature and duration on human adipose-derived stem cell viability for clinical use. Mol Med Rep, 2019; 19(3):2189–2201; doi: 10.3892/mmr.2019.9842

21.

Chen

, Yu

, Xue

, et al. Effects of storage solutions on the viability of human umbilical cord mesenchymal stem cells for transplantation. Cell Transplant, 2013; 22(6):1075–1086; doi: 10.3727/096368912X657602

22.

Al-Jaibaji

, Swioklo

, Shortt

, et al. Hypothermically stored adipose-derived mesenchymal stromal cell alginate bandages facilitate use of paracrine molecules for corneal wound healing. Int J Mol Sci, 2020; 21(16):5849; doi: 10.3390/ijms21165849

23.

Chen

, Wright

, Sahoo

, et al. A novel alternative to cryopreservation for the short-term storage of stem cells for use in cell therapy using alginate encapsulation. Tissue Eng Part C Methods, 2013; 19(7):568–576; doi: 10.1089/ten.TEC.2012.0489

24.

Tarusin

, Petrenko

, Semenchenko

, et al. Efficiency of the sucrose-based solution and UW solution for hypothermic storage of human mesenchymal stromal cells in suspension or within alginate microspheres. Probl Cryobiol Cryomedicine, 2015; 25(4):329–339; doi: 10.15407/cryo25.04.329

25.

Mahler

, Desille

, Frémond

, et al. Hypothermic storage and cryopreservation of hepatocytes: The protective effect of alginate gel against cell damages. Cell Transplant, 2003; 12(6):579–592; doi: 10.3727/000000003108747181

26.

Augst

, Kong

, Mooney

. Alginate hydrogels as biomaterials. Macromol Biosci, 2006; 6(8):623–633.

27.

Gryshkov

, Pogozhykh

, Zernetsch

, et al. Process engineering of high voltage alginate encapsulation of mesenchymal stem cells. Mater Sci Eng C Mater Biol Appl, 2014; 36:77–83; doi: 10.1016/j.msec.2013.11.048

28.

Liu

, Chang

, Wei

, et al. Comparison of the biological characteristics of mesenchymal stem cells derived from bone marrow and skin. Stem Cells Int, 2016; 2016:3658798; doi: 10.1155/2016/3658798

29.

Petrenko

, Rogulska

, Mutsenko

, et al. A sugar pretreatment as a new approach to the Me2SO- and xeno-free cryopreservation of human mesenchymal stromal cells. CryoLetters, 2014; 35:239–246.

30.

Tarusin

, Mazur

, Volkova

, et al. Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv. Encapsulation of mesenchymal stromal cells in alginate microspheres. Biotechnol Acta, 2016; 9(4):58–66; doi: 10.15407/biotech9.04.058

31.

Flaxman

. Cell identification in primary cell cultures from skin. In Vitro, 1974; 10(1–2):112–118; doi: 10.1007/BF02615344

32.

Dankberg

, Persidsky

. A test of granulocyte membrane integrity and phagocytic function. Cryobiology, 1976; 13(4):430–432.

33.

Fischer

, Jacobson

, Rose

, et al. Hematoxylin and eosin staining of tissue and cell sections. CSH Protoc, 2008; 2008:pdb.prot4986; doi: 10.1101/pdb.prot4986

34.

Costa

MHG

, McDevitt

, Cabral

JMS

, et al. Tridimensional configurations of human mesenchymal stem/stromal cells to enhance cell paracrine potential towards wound healing processes. J Biotechnol, 2017; 262:28–39; doi: 10.1016/j.jbiotec.2017.09.020

35.

Bauer

. Stem cell-based products in medicine: FDA regulatory considerations. Handbook of Stem Cells. 2004; 805–14; doi: 10.1016/B978-012436643-5/50163-2

36.

Sivandzade

, Bhalerao

, Cucullo

. Analysis of the mitochondrial membrane potential using the cationic JC-1 dye as a sensitive fluorescent probe. Bio Protoc, 2019; 9(1):e3128; doi: 10.21769/BioProtoc.3128

37.

Ding

, Liu

, et al. Cryopreservation with DMSO affects the DNA integrity, apoptosis, cell cycle and function of human bone mesenchymal stem cells. Cryobiology, 2024; 114:104847; doi: 10.1016/j.cryobiol.2024.104847

38.

Muraki

, Hirose

, Kotobuki

, et al. Assessment of viability and osteogenic ability of human mesenchymal stem cells after being stored in suspension for clinical transplantation. Tissue Eng, 2006; 12(6):1711–1719; doi: 10.1089/ten.2006.12.1711

39.

Pal

, Hanwate

, Totey

. Effect of holding time, temperature and different parenteral solutions on viability and functionality of adult bone marrow-derived mesenchymal stem cells before transplantation. J Tissue Eng Regen Med, 2008; 2(7):436–444; doi: 10.1002/term.109

40.

Lane

, Garls

, Mackintosh

, et al. Liquid storage of marrow stromal cells. Transfusion, 2009; 49(7):1471–1481; doi: 10.1111/j.1537-2995.2009.02138.x

41.

Sohn

, Heo

, Kim

, et al. Duration of in vitro storage affects the key stem cell features of human bone marrow-derived mesenchymal stromal cells for clinical transplantation. Cytotherapy, 2013; 15(4):460–466; doi: 10.1016/j.jcyt.2012.10.015

42.

, Wang

, Liu

, et al. Development and evaluation of a trehalose-contained solution formula to preserve hUC-MSCs at 4°C. J Cell Physiol, 2012; 227(3):879–884; doi: 10.1002/jcp.23066

43.

Petrenko

, Chudickova

, Vackova

, et al. Clinically relevant solution for the hypothermic storage and transportation of human multipotent mesenchymal stromal cells. Stem Cells Int, 2019; 2019:5909524; doi: 10.1155/2019/5909524

44.

Branco

, Tiago

, Laranjeira

, et al. Hypothermic preservation of adipose-derived mesenchymal stromal cells as a viable solution for the storage and distribution of cell therapy products. Bioengineering (Basel), 2022; 9(12):805; doi: 10.3390/bioengineering9120805

45.

de Souza

, Rosa

GDS

, Rossi

, et al. In vitro biological performance of alginate hydrogel capsules for stem cell delivery. Front Bioeng Biotechnol, 2021; 9:674581; doi: 10.3389/fbioe.2021.674581

46.

Klontzas

, Reakasame

, Silva

, et al. Oxidized alginate hydrogels with the GHK peptide enhance cord blood mesenchymal stem cell osteogenesis: A paradigm for metabolomics-based evaluation of biomaterial design. Acta Biomater, 2019; 88:224–240; doi: 10.1016/j.actbio.2019.02.017

47.

Passanha

, Gomes

, Piotrowska

, et al. Students of PRO3011. A comparative study of mesenchymal stem cells cultured as cell-only aggregates and in encapsulated hydrogels. J Tissue Eng Regen Med, 2022; 16(1):14–25; doi: 10.1002/term.3257

48.

Tümpel

, Rudolph

. Quiescence: Good and bad of stem cell aging. Trends Cell Biol, 2019; 29(8):672–685.

49.

Rumman

, Dhawan

, Kassem

. Concise review: Quiescence in adult stem cells: Biological significance and relevance to tissue regeneration. Stem Cells, 2015; 33(10):2903–2912; doi: 10.1002/stem.2056

50.

Fuller

, Petrenko

, Rodriguez

, et al. Biopreservation of hepatocytes: Current concepts on hypothermic preservation, cryopreservation, and vitrification. Cryo Letters, 2013; 34(4):432–452.

51.

Vreugdenhil

, Rankin

, Southard

. Cold storage sensitizes hepatocytes to oxidative stress injury. Transpl Int, 1997; 10(5):379–385; doi: 10.1007/s001470050074

52.

, Zhou

, Yin

, et al. Role of hypoxia in viability and endothelial differentiation potential of UC-MSCs and VEGF interference. Zhong Nan Da Xue Xue Bao Yi Xue Ban, 2013; 38(4):329–340; doi: 10.3969/j.issn.1672-7347.2013.04.001

53.

Thanuja

, Ranganath

, Srinivas

. Role of oxidative stress in the disruption of the endothelial apical junctional complex during corneal cold storage. J Ocul Pharmacol Ther, 2022; 38(10):664–681; doi: 10.1089/jop.2022.0082

54.

Zhou

, Liu

, Zhao

, et al. Natural melanin/alginate hydrogels achieve cardiac repair through ROS scavenging and macrophage polarization. Adv Sci (Weinh), 2021; 8(20):e2100505; doi: 10.1002/advs.202100505

55.

Guo

, Ma

, Shi

, et al. Alginate oligosaccharide prevents acute doxorubicin cardiotoxicity by suppressing oxidative stress and endoplasmic reticulum-mediated apoptosis. Mar Drugs, 2016; 14(12):231; doi: 10.3390/md14120231

56.

Zhao

, Han

, Wang

, et al. Alginate oligosaccharide protects endothelial cells against oxidative stress injury via integrin-α/FAK/PI3K signaling. Biotechnol Lett, 2020; 42(12):2749–2758; doi: 10.1007/s10529-020-03010-z