Encapsulation of human limbus-derived stromal/mesenchymal stem cells for biological preservation and transportation in extreme Indian conditions for clinical use

Human limbus-derived stromal/mesenchymal stem cells (hLMSC) can be one of the alternatives for the treatment of corneal scars. However, reliable methods of storing and transporting hLMSC remains a serious translational bottleneck. This study aimed to address these limitations by encapsulating hLMSC in alginate beads. Encapsulated hLMSC were kept in transit in a temperature-conditioned container at room temperature (RT) or stored at 4 °C for 3–5 days, which is the likely duration for transporting cells from bench-to-bedside. Non-encapsulated cells were used as controls. Post-storage, hLMSC were released from encapsulation, and viability-assessed cells were plated. After 48 and 96-hours in culture the survival, gene-expression and phenotypic characteristics of hLMSC were assessed. During transit, the container maintained an average temperature of 18.6 ± 1.8 °C, while the average ambient temperature was 31.4 ± 1.2 °C (p = 0.001). Encapsulated hLMSC under transit at RT were recovered with a higher viability (82.5 ± 0.9% and 76.9 ± 1.9%) after 3 (p = 0.0008) and 5-day storage (p = 0.0104) respectively as compared to 4 °C (65.2 ± 1.2% and 64.5 ± 0.8% respectively). Cells at RT also showed a trend towards greater survival-rates when cultured (74.3 ± 2.9% and 67.7 ± 9.8%) than cells stored at 4 °C (54.8 ± 9.04% and 52.4 ± 8.1%) after 3 and 5-days storage (p > 0.2). Non-encapsulated cells had negligible viability at RT and 4 °C. Encapsulated hLMSC (RT and 4 °C) maintained their characteristic phenotype (ABCG2, Pax6, CD90, p63-α, CD45, CD73, CD105, Vimentin and Collagen III). The findings of this study suggest that alginate encapsulation is an effective method of hLMSC preservation offering high cell viability over prolonged durations in transit at RT, therefore, potentially expanding the scope of cell-based therapy for corneal blindness.

The efficient shipping of cells from production facility to the site of application, while preserving the viability and quality of the cells, is very crucial 6 . Current methods of cryopreservation involve chemicals like dimethyl sulfoxide which itself is harmful to cells. Cryopreserving cells, in addition to being cost ineffective, has the drawbacks of decreased cell-viability 7 , impaired post-thaw function and reduced immunomodulatory properties 8 . The logistical complexity of transporting cells in their frozen state, accompanied by potential loss of function when used directly from the thaw, impedes the accessibility of cells for therapy at remote and rural sites. With increased regenerative research and the increased number of clinical trials, efficient transport (3-5 days in the current global scenario) of stem/progenitor cells from one institution to another where there is no GMP facility, is required. Autologous cells are an option but where autologous cells are not available, as in cases of bilateral eye damage/injury, allogeneic cells may be required. Cells, in general are transported using dry ice or liquid nitrogen modes, which is not cost effective, requires expedited shipping and packaging, suitable infrastructure, and specialised training for thawing and administration. In adverse events like a transportation delay, or change in temperature, cells can thaw and become unusable, or undergo stress affecting their viability and characteristic properties of cells 9 . One of the widely practiced alternatives to prevent this loss is encapsulating the cells in a biological matrix. Hypothermic preservation of encapsulated cells where cells are held in a state of suspended animation at temperatures below the normothermic range of 32 °C-37 °C also combats many of the issues associated with methods like cryopreservation 10 . Alginate is a natural polysaccharide exhibiting excellent biocompatibility and a popularly employed polymer for cell encapsulation. Alginate encapsulation has been reported to show more functionally robust spermatozoa 11 and oocytes 12 and to retain the morphological differentiation and adhesion abilities of the Neuroblastoma cells 13 . Recent studies have shown that encapsulating MSCs in alginate hydrogels could be a solution for problems associated with hypothermic storage through extending their preservation shelf life 10,14,15 . However, the reliability of alginate encapsulation has not been previously tested in geographies with high ambient temperatures or after long-distance transportation. This study aimed to test the reliability of alginate encapsulation for storing and transporting hLSMC at room temperature (RT) in temperate climatic conditions.

Methods and Materials
Study protocol and donor corneas. This study protocol was approved by the Institutional Review Board, LV Prasad Eye Institute, Hyderabad, India (LEC 05-18-081). Therapeutically accepted and serologically tested cadaveric donor corneas were obtained from Ramayamma International Eye Bank, LV Prasad Eye Institute, Hyderabad, India (http://www.lvpei.org/services/eyebank). Informed consent for using cadaveric corneas was obtained from the donors' next kin, by the Ramayamma International Eye Bank from where the cadaveric tissues were obtained. Experiments on the human tissue adhered to the declaration of Helsinki. All the experiments in the methodology were performed in triplicates.
Validating the insulated container for maintenance of hypothermic temperature. To have a reliable system that maintains a normalized range of temperatures irrespective of the extreme atmospheric temperatures, an insulated container with cooling packs (Polybox 7, Softbox Systems, India), pre-conditioned to maintain hypothermic temperatures of ≤30 °C, was assessed ( Supplementary Fig. S1). This assessment was done over a duration of 3-5 days, considering it the likely duration taken to transport cells. The internal temperature of the container and the ambient (atmospheric) temperature was recorded every 4 hours during this period.
Cell culture. The donor corneas were washed with 2% [vol/vol] Antibiotic-Antimycotic (15240062, Thermo Fisher, USA) in Phosphate Buffer Saline (PBS) (14190250, Thermo Fisher, USA) for 2 minutes, followed by another wash with PBS. Iris and endothelial layer were removed for better visibility of the limbus. Complete 360° limbal rims were isolated using a surgical blade in buffered saline and fragmented to minute pieces measuring 1-2 mm long. Tissue fragments were minced for 3-5 minutes using small, curved corneal scissors, in DMEM/ F12 media alone (BE04-687F/U1, Lonza, Switzerland). The minced limbal tissue was subjected to collagen digestion by adding 200 IU of reconstituted Collagenase-IV (17104019, Thermo Fisher, USA) in 1 mL of DMEM/F12 media. Tissue digestion was carried out by incubating the limbal tissue for 16 hours at 37 °C with 5% CO 2 in a humidified incubator.
Post 16-hour incubation, the enzymatic digestion was ceased by adding 2 mL of DMEM/F12 fortified with 2% fetal bovine serum (16000036, Thermo Fisher, USA). The enzyme-digested tissue fragments were washed and sedimented twice at 1000 rpm for 3 minutes, at room temperature (RT) in saline. 3 mL of complete media comprising of DMEM/F12 media fortified with 2% FBS, 1% [vol/vol] Antibiotic-Antimycotic, 10 ng/mL epidermal growth factor (PHG0311L, Thermo Fisher, USA) and 5 µg/mL insulin (12585014, Thermo Fisher, USA) was added to the pellet and kept in culture with culture medium being replaced every 2 days. Pure cultures of hLSMCs were obtained by subculturing. Subculturing was done upon 80-90% confluency. Passage 3 cells were used for all experiments post-quantification for viability using 0.4% Trypan Blue (15250061, Thermo Fisher, USA).

Encapsulation of hLSMCs.
A cell suspension of hLSMCs harvested from culture was mixed with sodium alginate solution supplied with BeadReady kit commercially available from Atelerix Ltd (UK) at a density of 2.5 × 10 6 cells/mL. The alginate-cell suspension concoction was slowly dropped into the calcium-chloride based gelation buffer (BeadReady kit) through a 21 1/2 G needle. These droplets of alginate-cell suspension concoction were allowed to stabilize for 8 minutes in the gelation buffer, making the beads polymerize and gelate (Fig. 1). Polymerized beads were washed with complete media and resuspended in 1 mL of fresh complete media.
Storage and transportation of the encapsulated hLSMCs. Vials with alginate-encapsulated cells in the form of polymerized beads were either refrigerated (4 °C; n = 5) or were kept under transit at RT (n = 5). The internal temperature of the container and the ambient (external) temperature was recorded every 4 hours, until 3-5 days (Fig. 1). The encapsulated cells were transported between three towns around Hyderabad, with a transport distance of ~528.67 (±64.2) kms. The vehicle used for the transit was a standard carrier vehicle. The external temperature outside the container was considered as control temperature. An equal number of the non-encapsulated cells were either stored or transported along, as above. All the packaging was done in controlled conditions. This experiment was performed in triplicates.
Release of the hLSMCs from encapsulation. Post transit, alginate beads encapsulating the cells were washed with PBS. They were added to 1.3 mL of dissolution buffer (trisodium citrate based), supplied with BeadReady kit and allowed to dissolve for 5 minutes with gentle agitation releasing the cells from the alginate beads. Cells suspended in the dissolution buffer were sedimented by centrifugation at 1500 rpm for 5 minutes. The sedimented cell pellet was resuspended in complete medium.

Quantifying the viable cells recovered. The number of viable (unstained) cells recovered from each vial
that were either stored at 4 °C or transported at RT was quantified using 0.4% Trypan blue solution and counted using a hemocytometer. Post quantification, the cells from the vials of same storage conditions (n = 5, each of RT and 4 °C), were pooled together. Pooled cells, along with non-encapsulated cells (cultured under standard culture conditions) as a control, were plated in equal numbers for further analysis of determining their relative survival, gene expression, and phenotypic biomarkers expression.
Determining the relative rate of survival using MTT assay. Post-release from 3-day and 5-day storage or transit, and their quantification for viability, the cells were plated in triplicates in a 12-well plate, at a density of 20,000 cells/cm 2 and cultured for 48 and 96 hours at 37 °C with 5% CO 2 in a humidified incubator. The relative survival rates of the cells against the control of non-encapsulated (cultured under standard culture conditions) cells were assessed using MTT reagent (M6494, Thermo Fisher, USA). Each well was added with 200 µL of 0.25 mg/mL MTT reagent in culture medium devoid of FBS and incubated for 1 hour at 37 °C in 5% CO 2 chamber. The formazon crystals were solubilized in 200 µL of Dimethyl Sulfoxide (D2650, Sigma Aldrich, USA) for 5 minutes at 37 °C in 5% CO 2 chamber. The concentration was determined by reading the absorbance in duplicates at 570 nm using a spectrophotometer, against a blank.

Assessment of the phenotypic marker expression. Encapsulated cells that were either transported at
RT or were under storage at 4 °C for 3-5 days were released and quantified for viability. Cells were cultured on coverslips in 12-well culture plates at a density of 20,000 cells/cm 2 at 37 °C with 5% CO 2 in a humidified incubator for 48 hours. These cells were assessed for the expression of characteristic biomarkers of the hLMSC phenotype. Cultured cells were washed with PBS and fixed using 4% paraformaldehyde in PBS, for 20 minutes, followed by a 10-minute wash in PBS, twice. The cells were permeabilized using 0.03% [vol/vol] Triton-X in PBS, followed by two 5-minute washes in PBS. Cells were incubated for 1 hour with 2.5% BSA in PBS, to block the non-specific www.nature.com/scientificreports www.nature.com/scientificreports/ protein-protein interactions. All the incubations were carried out at RT in moist conditions. The blocking solution was removed and cells were incubated for 2 hours with primary antibodies in 100 µL of 1% BSA in PBS. Quantification of the gene expression using real-time PCR. One million cells of each storage category after their release from encapsulation were used for quantifying the gene expression. Freshly lysed trypsinized cells from the culture were used as the control. Total RNA was isolated using Trizol (15596018, Thermo Fisher, USA) method and converted to cDNA using the Superscript-III (1808051, Thermo Fisher, USA) at 1 µg/ µL of RNA per 20 µL reaction mix. The synthesized cDNA was subjected to real-time PCR, using Maxima SYBR Green kit (K0221, Thermo Fisher, USA) with 200 ng template per 25 µL reaction mix. The reaction was carried out in a detection system (Applied Biosystems, USA). Reactions were run in duplicates. GAPDH was used as a housekeeping gene in these experiments. The gene expression data were normalized to control the variability in expression levels to the geometric mean of the housekeeping gene. The data was analysed using the 2 −ΔΔCT method. The primer sequences are listed in the Table 1.

Statistical analysis. Statistical analyses were done using the GraphPad software (GraphPad Software, San
Diego, CA, http://www.graphpad.com). Comparisons were made using Mann-Whitney U test for non-parametric data. The data is presented as mean values ± SD, obtained from 3-10 independent experiments performed. Values of p < 0.05 were considered to be significant. *p ≤ 0.05, **p ≤ 0.001. Values of p > 0.05 were considered insignificant and were represented with #.

Results
Maintenance of hypothermic temperatures in the pre-conditioned container. The container maintained an average temperature of 18.62 ± 1.82 °C (range: 13.91 °C to 27.52 °C) where the average ambient temperature was 31.43 ± 1.2 °C (range: 28.85 °C to 38.40 °C) over a duration of 3-5 days (Fig. 2). This experiment was repeated (n = 10) and the data was statistically significant (p < 0.0001). The container had maintained the hypothermic range of temperatures consistently over a period of varying seasons and weather across the year.
Effect of temperature on viable cell recovery. The temperature in the storage conditions had an insignificant effect on the recovery of viable cells from encapsulation. Encapsulated cells recovered after 3-day transit at RT had an average viable recovery of 82.45 ± 0.87% (n = 3) cells while the cells stored at 4 °C had 65.19 ± 1.19% (n = 3, p = 0.0008) viability. After 5-day transit at RT, encapsulated cells had 76.96 ± 1.98% (n = 3) and cells stored at 4 °C had 64.45 ± 0.81% (n = 3, p = 0.0104) of viable cell recovery (Fig. 3). The non-encapsulated cells stored at RT did not show more than 1% viability during both 3-day and 5-day transit. Non-encapsulated cells stored at 4 °C showed a mean recovery of 5.33% on 3-day storage and up to 4% after 5-day storage.  (Fig. 4). On the other hand, the encapsulated cells stored under refrigerated conditions for 3-days, have shown attachment of about 39.67 ± 5.32% after 48 hours and which increased to 54.8 ± 9.04% after 96 hours. Upon 5-day refrigeration, the cell attachment was 43.77 ± 3.53% after 48 hours and 52.35 ± 8.07% after 96 hours.
Phenotypic expression of the biomarkers. Encapsulated cells under transit at RT have shown the similar (default) phenotype with the control group of cells, during both 3-day and 5-day transit. Encapsulated cells under storage at 4 °C showed expression of ABCG2 after 3-day storage but not at 5-day refrigeration (Figs 5 and  6). The expression of the rest of the biomarkers by both RT and 4 °C groups was similar to the control cells, showing the positive expression of Pax6 + and stem cell markers (p63-α + , ABCG2 + ) and the biomarkers for mesenchymal origin (VIM + , CD105 + , CD90 + , CD45 − ) and the other surface biomarkers Col-III + , and CD73 + . Although HLA-DR is considered negative marker for the mesenchymal origin, we have found this marker to be positively   (Table 2).

Quantifying the gene expression (RT-PCR). Although encapsulated cells stored at RT and 4 °C showed
higher levels of PAX-6, p63-α, and CD90 expression as compared to the control group, these differences were not statistically significant (Fig. 7, p > 0.11).

Discussion
This study aimed to evaluate the efficacy of alginate encapsulation in maintaining the viability and properties of hLMSC while being stored and transported at RT in a real-life ground-transportation scenario. The study found that while non-encapsulated cells had negligible viability at RT and 4 °C, encapsulated hLMSC (RT and 4 °C) maintained high viability, had good survival in culture and retained adequate phenotype expression. The phenotypic assessment of the encapsulated cells in comparison with control groups showing the number of cells positive for a given biomarker is given in Table 2. A similar trend of the percentage of cells expressing a biomarker was observed. We have found positive expression of HLA-DR in all the groups of cells. Many earlier studies have shown similar findings of the positive expression of HLA-DR in the normal cornea towards periphery and the limbus [17][18][19] . The findings of this study suggest that alginate encapsulation is an effective method of hLMSC preservation and transport at RT for up to 3 to 5 days, which would allow these cells to be shipped to remote locations and therefore, potentially expand the scope of cell-based therapy for corneal blindness.
Corneal stromal stem cells and more recently hLMSC have been studied for their ability to restore corneal transparency 3 through corneal stromal regeneration 20 . The therapeutic potential of these cells for treating various corneal pathologies is currently being explored in clinical trials and the initial reports have shown enhancement in visual parameters and corneal epithelization, neovascularization and clarity 4,21,22 . These cells may eventually evolve into a simpler non-invasive alternative to corneal transplantation, thereby reducing the global demand for donor corneas. Further expansion of this therapeutic advancement is hindered by the bottlenecks of lacking proper preservation and transport methods towards the delivery of these cells without affecting their characteristic properties. The maintenance of appropriate temperature is a crucial and integral factor for optimal shelf life of the cells 23 . Despite the ambient temperature fluctuations between 28.9 to 38.4 °C, not only was the insulated container able to maintain significantly lower temperatures of 13.9 to 27.5 °C, but alginate encapsulation also allowed most cells to survive while in transit. The proportion of the encapsulated cells that were lost in the transit, may be considered to have undergone apoptosis. However, without encapsulation almost all cells perished within the same amount of time. Ability to transport cells at RT circumvents the usage of dry ice, which is currently categorized as restricted item for airborne transport (https://www.fedex.com/in/domestic/services/regulatoryguidelines.html) and of any expensive equipment required to maintain chilled temperature during shipping. This would potentially translate into significantly lower costs for cell storage and transportation.  www.nature.com/scientificreports www.nature.com/scientificreports/ experimental temperature ranges tested previously were 22-25 °C 16 or 11-23 °C 10 in controlled laboratory set-ups, in this study the external ambient temperature ranged from 28.9 to 38.4 °C in real-life conditions. These results imply that it may be possible to send the alginate encapsulated cells to remote locations for their application using ground transportation, which would significantly lower the shipping costs involved. The remote and rural areas, Figure 6. Quantification of the gene expression using real-time PCR. Immunostaining of the encapsulated hLSMCs under transit for 5 days: Alginate encapsulated hLSMCs stored at 4 °C did not show expression of the stem-cell (ABCG2 − ). The RT group cells have showed similar phenotype as the control group (ABCG2 + , Pax6 + p63-α + , VIM + , CD90 + , CD105 + , CD45 − , HLADR + , Col-III + , and CD73 + ). Blue: DAPI, nuclear stain. Scale: 100 µM.
www.nature.com/scientificreports www.nature.com/scientificreports/ by having equipped with one centrifuge and a pipette, shall be able to release the encapsulated cells, without the necessity of having a cell culture facility. Additionally, all the reagents and procedures employed in the process of cell encapsulation are FDA approved. This would ease the regulatory constraints on the clinical translation and expansion of the technique 26 . However, this study is limited by lacking serum free culture methods and the study of therapeutic properties of the encapsulated cells, which are underway in the further phase of this study.
In conclusion, this study aimed to test the reliability of alginate encapsulation for storing and transporting hLSMC at RT in temperate climatic conditions and the findings of this study suggest that alginate encapsulation is an effective method of hLMSC preservation offering high cell viability over prolonged durations in real-life transit conditions. The simplicity of the encapsulation process combined with the cost-effectiveness of ground-transportation makes alginate encapsulation an attractive option for furthering the scope and scale of cell-based therapy for corneal blindness particularly in the developing world.