In Vitro Culture Expansion and Characterization of Buccal Mucosal Epithelial Cells for Tissue Engineering Applications in Urethral Stricture After Transportation Using a Thermoreversible Gelation Polymer

Abstract

Introduction:

The transportation of tissues from hospitals to clinical laboratories for cell therapy is an essential component of regenerative medicine. Previously, we used laboratory-cultured mucosal cells from buccal epithelium expanded and encapsulated using a scaffold-hybrid approach to the urethral stricture (BEES-HAUS) procedure. In this study, to improve the outcomes, we compared the thermoreversible gelation polymer (TGP) transportation procedure with conventional culture methods, and reported its advantages.

Methods:

Human buccal mucosal tissues in Phase I of the study were transported in Euro-Collins solution (ECS) and the cells obtained were cultured in two-dimensional (2D) Dulbecco's modified Eagle's medium (DMEM), CnT-Prime epithelial 2D differentiation medium (CnT-PR), and a three-dimensional (3D)—TGP scaffold. In Phase II, tissues were transported in a TGP cocktail and the ECS. The cells were cultured in 2D-DMEM and 3D-TGP, quantified, and characterized by immunohistochemistry.

Results:

The cells in 3D-TGP culture maintained epithelial morphology in a better manner compared with 2D-DMEM, in which they developed fibroblast-like morphology. The TGP-transported cells grew rapidly. Immunohistochemical analysis results for AE1/AE3, EGFR, integrin-β1, p63, and p75 were intensely positive in 3D-TGP.

Conclusion:

The TGP-based cocktail used in human buccal tissue transportation yielded cells with better morphology maintenance. The TGP scaffold provides an optimal in vitro environment wherein epithelial cells better maintain their native phenotype compared to those cultured through conventional methods. These results suggest using TGP for the transportation and culture of human buccal tissues for clinical applications. In addition, the use of a TGP-based cocktail for the transport of other tissues for regenerative medicine applications is worth further analysis.

Color images are available online

Introduction

Cells, namely progenitor cells and stem cells, are essential components of regenerative therapies and need to be processed in vitro or cultured for different durations depending on the application. Transportation of tissue samples from the place of harvest, that is, a hospital, to the laboratory for processing requires highly efficient preservation, storage, and transportation methods to preserve the viability of the cells in the tissue samples outside their natural physiological environment and minimize the effects of the harsh conditions faced during transportation.

Methods for organ preservation are well established, and two types of preservation media are employed: intracellular fluid (ICF)-type preservation solutions and extracellular fluid (ECF)-type preservation solutions.¹ Euro-Collins solution (ECS) and the University of Wisconsin solution are examples of ICF-type preservation solutions. These solutions protect the intracellular space from ischemic injury due to high potassium and low sodium content.

The difficulty in overcoming the risk of hyperkalemia-induced pulmonary vasoconstriction with ICF-type solutions has led to the development of ECF-type (low potassium) solutions such as histidine–tryptophan–ketoglutarate, Celsior, Perfadex Papworth, and Plegisol.² For the preservation of tissue and cells, phosphate-buffered saline (PBS), Dulbecco's modified Eagle's medium (DMEM), and ECS remain the most commonly employed media depending on the required duration of preservation.¹

Maintaining the temperature at 4°C or cool preservation is a mandatory requirement when these solutions are used. Rao et al.³ investigated the transportation of cadaveric donor corneal endothelial tissue using a thermoreversible gelation polymer (TGP), which was able to preserve cell viability for over 72 hours at varying temperatures, without the need for cool preservation.³

Human corneal endothelial progenitor cells (HCEPs) were cultured from transported corneal endothelial tissue and used for allogeneic transplantation to treat bullous keratopathy in a pilot clinical study.⁴ We have previously reported a human clinical pilot study on the buccal epithelium expanded and encapsulated using a scaffold-hybrid approach to the urethral stricture (BEES-HAUS) procedure, which is a cell therapy method using in vitro-expanded autologous buccal mucosal cells.⁵ During the process of investigating the BEES-HAUS approach in a multicenter randomized clinical study in Japan, we reported the standardization of the culture method earlier.⁶

Then, we began identifying and standardizing potential solutions to existing obstacles in tissue transportation from the place of biopsy harvest to the laboratory. We investigated the approach presented by Rao et al.³ using TGP to transport buccal tissue biopsies, which we describe in this article.

Materials and Methods

The study was done in two phases:

In Phase I, buccal tissue samples were transported only in the ECS and cultured in three groups using explant culture.

Group I: Two-dimensional (2D) DMEM (Gibco BRL, Gaithersburg, MD) without scaffold (n = 3).

Group II: CnT-Prime epithelial 2D differentiation medium (CnT-PR) (CellnTec, Switzerland) without scaffold (n = 3).

Group III: Three-dimensional (3D) using TGP scaffold (n = 3).

In Phase II, we compared buccal tissue transportation in TGP (n = 3) reconstituted with DMEM at 25°C with transportation in ECS (n = 3) at 4°C, at 4 hours, followed by the culturing of tissues in two groups: (1) 2D in DMEM without scaffold and (2) 3D using TGP scaffold.

Buccal mucosal biopsies

The study was performed in accordance with the Declaration of Helsinki, as revised in 2013, following all guidelines and regulations. The study was approved by the Ethics Committee of the National Defense Medical College, Japan (Ethics Committee Approval number: 4154; April 9, 2020). Human buccal tissue samples were obtained from adult patients undergoing biopsy for buccal mucosal graft urethroplasty. Informed consent was obtained for the collection of all samples, which were redundant after use in surgery. The size of the donor tissues varied among patients.

Preparation of TGP

Lyophilized TGP vials (1 g) were provided by M/s GN Corporation (Mebiol Inc., Japan). Japan). The sol–gel transition temperature of the TGP used in this study was 20°C. Thus, lyophilized TGP becomes gelatinized above 20°C, while it becomes liquefied at temperatures below 20°C.⁷ Nine milliliters of DMEM/F12 medium (Gibco BRL) was added to the 1-g TGP vial for reconstitution when the gel dissolves into the medium and kept at 4°C overnight.⁸ The reconstituted TGP was incubated at 4°C until use.

Transportation

For transportation in the ECS groups, three buccal tissue samples (n = 3) were immersed in vials containing ECS, and the vials were placed in a box with temperature-controlled packs and a temperature logger to maintain the temperature at 4°C. For transportation in the TGP group, three buccal tissue samples (n = 3) were placed in vials containing TGP cooled to its liquefying temperature.³ TGP was then allowed to warm to the room temperature, (≥20°C) for gelatinization, and then placed in a box with temperature controlling packs and a temperature logger to maintain the temperature above 20°C during transportation. The duration of transportation from the hospital to the cell-processing laboratory was 4 hours.

Cell processing and culture

Upon reaching the laboratory, in Phases I and II, the tissue samples were removed from the ECS and TGP vials using sterile tweezers and washed with 1% penicillin–streptomycin (P/S), 50 μg/mL gentamicin (GM), and 0.25 μg/mL amphotericin B (Amp B) in Dulbecco's PBS. Each tissue sample was cut into tiny pieces and immersed in a few drops of phosphate-buffered solution to prevent dehydration. The tissues were then divided into two portions: one portion was seeded into a TGP scaffold using a TGP scaffold-based culture methodology, whereas the other was cultured without the TGP scaffold.^5,6

In both phases, the culture medium used in 2D-DMEM contained 10% autologous serum obtained from the peripheral blood of the patient from whom the biopsy was taken, 1% P/S, 50 μg/mL GM, 0.25 μg/mL Amp B, 5 μg/mL human insulin (91077C; Sigma, USA), and 10 ng/mL human epidermal growth factor (hEGF), whereas the culture medium in the other group was 2D CnT-PR.

For the TGP culture, 1 mL of TGP was reconstituted in 9 mL of DMEM. A drop of TGP-reconstituted DMEM (TGP-DMEM) was placed in the center of a 12-well tissue culture plate and solidified at 37°C. Each tissue piece was carefully placed into the well plate, and a drop of TGP-DMEM was placed over the tissue piece/cell drop, resulting in the tissue pieces or cells being sandwiched by TGP-DMEM. The culture medium (1% P/S, 50 μg/mL GM, 0.25 μg/mL Amp B, 5 μg/mL human insulin, and 10 ng/mL hEGF)⁹ was then added. This group was termed 3D-TGP. All tissue culture plates were placed in a humidified CO₂ incubator at 37°C. The maximum culture duration was 29 days.

After culturing, cells from all the culture groups were harvested. For the 2D cultures, trypsin was used to harvest the cells, whereas in the 3D-TGP group, the tissue culture plates were cooled down to a temperature of 4°C for 20 minutes and then centrifuged to collect the pellet and harvest the cells.

Immunohistochemistry

The cells obtained from the different groups were fixed using formalin. The formalin-fixed paraffin-embedded cells were sectioned at 4 μm thickness. Immunohistochemical staining (IHC) for AE1/AE3, EGFR, integrin-β1, p63, and p75 was performed using a Ventana Benchmark XT automated slide staining system (Ventana Medical Systems, USA).

The sections were deparaffinized, pretreated with cell conditioning 1 (Ventana Medical Systems), reacted with primary antibodies for 32 minutes at room temperature, visualized with the iView DAB Detection Kit (Ventana Medical Systems), and counterstained with Hematoxylin (Ventana Medical Systems) and bluing reagent (Ventana Medical Systems). The antibodies were diluted to 1:1 and used with the iVIEW DAB Detection Kit (Ventana Medical Systems) and an Endogenous Biotin-Blocking Kit (Ventana Medical Systems).

The following simple qualitative scoring system at 100 × magnification in 10 random fields was employed for each image after IHC for the different markers (AE1/AE3, EGFR, integrin-β1, p63, and p75):

“+++” score was given when 70%–95% of the cells stained positive;

“++” score was given when 50%–69% of the cells stained positive;

“+” score was given when 10%–49% of the cells stained positive;

“−” score when <10% of the cells stained positive or when no staining was visibly observed.

Statistical analysis

All data were analyzed using the Excel statistics package analysis software (Microsoft Office Excel^®); repeated measures analysis of variance, including Tukey's honestly significant difference was used; p-values <0.05 were considered significant.

Results

The average cell count (mean ± standard deviation [SD]) obtained after explant-culture in Phase I for ECS-transported 2D-DMEM was 0.23 ± 0.16 million cells, whereas for 3D-TGP, it was 0.16 ± 0.02 million cells. The differences between the groups were not statistically significant (p = 0.225). The number of cells in the CnT-PR was too small to be counted.

The average cell count (mean ± SD) obtained in Phase II for ECS-transported 2D-DMEM and 3D-TGP was 0.12 ± 0.007 million and was 0.44 ± 0.007 million, respectively. In TGP-transported specimens, the average cell counts obtained in 2D-DMEM and 3D-TGP were 0.32 ± 0.013 million and 0.51 ± 0.22 million, respectively (Fig. 1). The differences between the groups were not statistically significant (p = 0.068332).

FIG. 1.

Average cell count in different groups after culture shows a higher count in TGP-transported and TGP-cultured group (TGP-3D) than in other groups. 2D, two-dimensional; 3D, three-dimensional; TGP, thermoreversible gelation polymer.

In Phase I, cells in 3D-TGP grew well with epithelial morphology, cells in 2D-DMEM grew slowly with fibroblast morphology, and in the CnT-PR group, cell proliferation was low (Fig. 1). In Phase II, ECS-transported specimens in 2D-DMEM grew slowly with fibroblast morphology, whereas in TGP, although the growth was slow, the cell number was relatively high. TGP-transported cells in 2D grew slowly, but in 3D (TGP), the growth was rapid and cells were healthier with epithelial polygonal morphology maintained (Fig. 2). IHC for markers AE1/AE3, EGFR, integrin-β1, p63, and p75 was positive, with more intense staining in the 3D-TGP cultured cells (+++ AE1/AE3 and p63; ++ for EGFR and integrin-β1) compared with that in the 2D-DMEM group (+ AE1/AE3, p63; EGFR and integrin-β1) (Fig. 3).

FIG. 2.

Cells derived from TGP-transported tissue grew better with native polygonal epithelial morphology maintained in 3D-TGP culture compared with the ECS-transported groups. Cell clusters were also observed in TGP cultures (arrows) (10 × ). CnT-PR, CnT-Prime epithelial 2D differentiation medium; DMEM, Dulbecco's modified Eagle's medium; ECS, Euro-Collins solution. Color images are available online.

FIG. 3.

Immunohistochemical staining: the presence of more intensely stained cells (+++ AE1/AE3 and p63; ++ for EGFR and integrin-β1) in 3D-TGP cultured cells than in 2D grown cells (+) wherein, AE1/AE3, EGFR, and integrin-β1 are indicative of epithelial phenotype, whereas p63+ cells are indicative of progenitors. Color images are available online.

Discussion

While the media used for organ preservation and transportation have been well established with ongoing technological advancements, transportation of tissue specimens from the place of harvest to the laboratory is usually done using a culture medium or organ preservation medium like ECS at 4°C.¹ Rao et al.³ and Senthilkumar et al.⁹ have shown that a TGP-based cocktail allows the transportation and storage of sensitive tissues such as human corneal endothelium and human retinal pigment epithelium for over 72 hours without needing cool preservation.^3,11

Human corneal endothelial tissue, which contains highly sensitive cells, was safely transported using the TGP scaffold and used to derive HCEPs. These in vitro culture-expanded HCEPs have been used for transplantation in human patients in a pilot study with positive outcomes.⁴ Since highly sensitive tissues have been transported using this optimal polymer encapsulated tissue transport for regenerative applications, cell therapy and transplant (OPTRACT) methodology,¹² this technology is also extendable to other cells and tissues.

We previously reported the endoscopic-based buccal epithelium expanded and encapsulated in a scaffold-hybrid approach to the urethral stricture (BEES-HAUS) technique⁵ in which six male patients with bulbar urethral stricture were treated with autologous buccal mucosal epithelial cells expanded in vitro using the 3D-TGP culture-based methodology. To expand the technique to a randomized multicenter clinical trial, we began standardizing the transport methodology, as the PBS used in the earlier study allows a limited time for transportation and requires temperature maintenance for cold preservation.⁵

We then studied transportation in the ECS in Phase I, as ECS is one of the most commonly used, low-cost organ preservation solutions.^1,13 Although we were able to retrieve viable cells after transportation and were able to grow a relatively better quality of cells in 3D-TGP compared with 2D-DMEM culture, ECS allowed a limited transportation time and cool preservation was a mandatory requirement.¹ Following Rao et al.³ and Senthilkumar et al.,⁹ we used TGP in transportation, which does not require cool preservation, making it useful for transportation of cells and tissues in developing nations where cold-chain management is still difficult.¹⁴

Hence, in Phase II, we compared ease of transportation using TGP with that using ECS. We were able to retrieve a slightly higher number of cells in TGP-transported tissue than in ECS-transported tissues, especially in 3D-TGP-cultured explants, with TGP-cultured cells exhibiting native polygonal epithelial phenotype along with some cells forming clusters indicative of progenitor populations. This was confirmed by immunohistochemistry, wherein AE1/AE3, EGFR, and integrin-β1 positivity were indicative of an epithelial phenotype, whereas p63 and p75 positivity was indicative of epithelial progenitors. All these markers stained more intensely in the 3D-TGP-cultured cells.

Since CnT-prime epithelial 2D differentiation medium (2D CnT-PR) is a common animal component-free culture medium for isolation and expansion of epithelial cells,^14,15 we compared it with DMEM in Phase I. However, since cells did not grow well (Fig. 1), we used only DMEM for the cell culture in Phase II of the study.

There are a few limitations to this study. In Phase II, the source tissue was divided for transportation into the ECS and TGP groups by gross assessment of tissue size and weight, but a slight initial variation in the number of cells among the tissue samples transported in the ECS and TGP is probable. Another limitation was the small number of samples and the fact we did not culture the samples by digestion culture, following the example in Vaddi et al.⁵ We attempted explant culture based on the observations by Sudha et al.⁸ that corneal epithelial tissue as explants sandwiched in TGP grows healthily. Since the buccal and corneal epithelium share similar cell surface markers,¹⁵ we used this methodology to investigate whether explant culture yields a higher number of cells.

However, the cell count obtained was less than that in our earlier study of BEES-HAUS,^5,7 in which we used a digestion culture. We used digestion culture in BEES-HAUS as conventional buccal epithelial cultures used in human clinical transplantation predominantly employ digestion cultures.¹⁶ As it was our first experiment with buccal mucosal cells, we compared our protocol with the conventional protocol followed for epithelial cells reported in the literature.¹⁷ We are now in the process of validating these findings through further experimentation.

Conclusions

Transportation of human buccal tissue specimens in a TGP-based cocktail yielded a significantly better quality of cells than transportation in the ECS. Subsequent in vitro culture of buccal mucosal tissue as explants derived from the TGP-transported tissue in TGP yielded more prolific cells with healthier epithelial phenotype maintenance and more intense positivity for epithelial and progenitor markers than did the DMEM-cultured or CnT-PR-cultured cells. These results suggest that TGP-based methods are favorable for the transportation of human buccal tissue and its in vitro culture for clinical applications in cell therapy for the urethral stricture, such as the previously reported BEES-HAUS method.⁵ An extension of this technology to the transport of other cells and tissues for regenerative medicine applications is also recommended.

Footnotes

Authors' Contributions

A.H., V.S., and S.J.K.A. contributed to conception and design of the study. K.O., M.S., Y.M., T.K., R.S., and M.B. helped in data collection and analysis. SA drafted the article. A.H., M.T., M.I., S.K., H.Y., and S.J.K.A. performed critical revision of the article. All the authors read, and approved the submitted version.

Acknowledgments

The authors thank Dr. Fumihiro Ijima of Hasumi International Research Foundation, Asagaya, Tokyo, Japan for his assistance with the cell culture work described in the article. Ms. Eiko Amemiya, Yamanashi University, Japan for her assistance with literature collection. Mr. Rajmohan Mathaiyan and Mr. Ramalingam Karthik from NCRM, India for their assistance with the data collection and transportation protocols. Dr. Senthilkumar Preethy from NCRM, India for her assistance in drafting the article. Loyola ICAM College of Engineering Technology (LICET), Chennai, India for their support to our research work. M/s Department of Clinical Research, Yamanashi University, Japan for their assistance with the publication of the article.

Ethics Approval

The Institutional Ethics Committee of the National Defense Medical College (NDMC) approved the study. The Ethics Committee Approval number: 4154 (April 9, 2020).

Availability of Data and Materials

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

Author Disclosure Statement

S.K. is an employee of Edogawa Hospital, Japan and is an applicant/inventor to several patents on biomaterials and cell culture methodologies, some of them described in this article. H.Y. is an employee of Mebiol Inc., and an applicant to several patents on TGP and its applications. S.J.K.A. is a shareholder in GN Corporation Co. Ltd., Japan and is an applicant/inventor to several patents on biomaterials and cell culture methodologies, some of them described in this article.

Funding Information

No external funding was obtained for the study.

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