Preclinical assessment of transplantable human nasal mucosal epithelial cell sheets.

INTRODUCTION: We previously reported a new cell transplantation therapy for patients with intractable otitis media using autologous nasal mucosal epithelial cell sheets, manufactured using temperature-responsive cell culture inserts. The current study aimed to verify whether the transplantable cell sheets could be manufactured for application in clinical trials, according to standard operational procedures (SOP), in a cell processing facility (CPF).
METHODS: Human nasal mucosal epithelial cells from four volunteer donors were aseptically cultured and transplantable cell sheets successfully manufactured, with reproducibility, using temperature-responsive cell culture inserts in the CPF. During the manufacture of cell sheets, the CPF environment was confirmed to be aseptic by sterilization tests. Purity of the cell sheets was confirmed by histological analysis and flow cytometry. Both safety and quality of the human nasal mucosal epithelial cell sheets were validated.
RESULTS: The cultured and manipulated human nasal mucosal epithelial cells showed no evidence of malignant transformation in vitro. The study confirmed the safety and suitability of the manufactured human nasal mucosal epithelial cell sheets for use in clinical trials.
CONCLUSIONS: The results led to the establishment of a coherent system in which transplantation could be achieved smoothly.

Introduction

Cell therapy is of great concern in regenerative medicine. We previously reported a new cell transplantation therapy by applying autologous nasal mucosal epithelial cell sheets to damaged middle ear for postoperative mucosal regeneration, using temperature-responsive cell culture inserts [1]. Using this new therapy, we could successfully treat patients with intractable otitis media in a clinical study [2]. Cells cultured on temperature-responsive cell culture inserts can be harvested as a contiguous sheet by simply lowering the culture temperature without using proteolytic enzymes or other biomaterials [[3], [4], [5]]. Cell sheet technology using temperature-responsive cell culture inserts has been clinically applied to various tissue regeneration processes till date, such as for corneal epithelium [6], esophagus [7], cardiac muscle [8], and cartilage [9]. Stable manipulation of cells and manufacture of cell sheets would be of great importance in clinical use. Therefore, in this study, we established a coherent system in which the process of transplantation could be smoothly conducted.

We created standard operational procedures (SOP) that documented the methods of cultivation and manufacture of human nasal mucosal epithelial cell sheets using temperature-responsive cell culture inserts, as well as the methods for validating the quality of manufactured cell sheets. In this preclinical study, we verified whether transplantable cell sheets could be manufactured using human nasal mucosa collected from 4 healthy volunteers in a cell processing facility (CPF), whose environment is kept clean under controlled temperature and pressure as per the SOP. The manufactured human nasal mucosal epithelial cell sheets were validated to secure their adequacy for transplantation into human middle ear. Environment of the CPF was also confirmed to be aseptic during this study. Overall, we demonstrated the safety and quality of human epithelial cell sheets in this preclinical study.

Methods

Validation test of the CPF

All procedures for the manufacture of cell sheets were carried out in a good manufacturing practice (GMP)-compliant CPF located at Jikei University School of Medicine (Fig. 1). Clean culture environment was ensured in the CPF, and the temperature and pressure in each room were well-controlled. In the cell culture room, the double dressing gown system was used following GMP. Sterilization tests were performed 12 times over 6 months to ensure aseptic environment in the CPF. Floor samples of the CPF were inoculated in agar medium to detect adherent bacteria and fungi.

Fig. 1

Design of the good manufacturing practice-compliant cell processing facility (CPF). In a good manufacturing practice-compliant CPF, human nasal mucosal epithelial cells were manipulated and subsequently, cell sheets were manufactured. Environment of each room in the CPF is represented in terms of atmospheric pressure (Pa) and cleanliness. Number of aerosol particles in class 103 and 104 areas were kept less than 103 and 104 particles/ft3, respectively.

Preparation of culture medium

We used the keratinocyte culture medium (KCM) for cell culture and manufacture of cell sheets. KCM comprised a basal mixture of three parts Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher Scientific, MA, USA) and one part Ham's F-12 nutrient mixture (Gibco), containing 5% autologous human serum of each patient, insulin (140.0 mU/mL, Humulin, Novo Nordisk, Bagsvaerd, Denmark), triiodothyronine (2.0 nM, MP Biomedicals, CA, USA), saxizon (0.3 μM, Takeda Yakuhin Kogyo, Osaka, Japan), epidermal growth factor (0.2 μM, Higeta-Shoyu, Tokyo, Japan), cholera toxin (1.0 nM, Wako Pure Chemicals, Osaka, Japan), penicillin (100 U/mL, Wako Pure Chemicals), streptomycin (69 μM, Wako Pure Chemicals), and amphotericin B (0.4 μg/mL, Bristol-Myers Squibb, NY, USA).

Isolation of nasal mucosal tissue

This study was formally approved by the ethics committee of Jikei University School of Medicine (approval number: 21-074). Both verbal and written informed consents were obtained from all volunteers when human nasal mucosal tissues were used. The nasal cavity was often observed first through nasal endoscopy, and all volunteers were confirmed to have no pre-existing nasal disease (Fig. 1A). Local anesthesia of the inferior nasal concha membrane in the part to be excised was performed by xylocaine (AstraZeneca, Osaka, Japan). Approximately 10-mm (i.d.) biopsies of nasal mucosa were collected from inferior nasal concha using surgical scissors under nasal endoscopy (Fig. 2B and C). Hemostasis was done using bipolar coagulation forceps and confirmed thereafter in the excised part (Fig. 2D). The harvested nasal mucosa was then incubated in DMEM containing penicillin (100 U/mL) and streptomycin sulfate (69 μM), and transferred to the CPF.

Fig. 2

Isolation of nasal mucosal tissue. (A) Using nasal endoscopy, the nasal cavity was observed first, and no pre-existing nasal disease was confirmed. (B) The inferior nasal concha mucosa was appropriately excised based on nasal endoscopy. (C) Approximately 10-mm (i.d.) tissue of nasal mucosa was harvested. Bars indicate 10 mm. (D) Hemostasis was confirmed and no re-bleeding was found in the excised parts of all four volunteer donors. (E) The harvested nasal mucosa was moved to the CPF, and all procedures to prepare human nasal mucosal epithelial cells and fabricate cell sheets were performed under asepsis in the CPF.

Manufacture of human nasal mucosal epithelial cell sheets

According to SOP, all procedures were performed aseptically in the CPF (Fig. 2E). Biopsy specimens were sterilized twice with povidone iodine and washed with DMEM containing penicillin (100 U/mL) and streptomycin sulfate (69 mM). The nasal mucosal epithelia of sterilized nasal mucosal tissue were separated from the underlying sub-epithelium using a surgical knife. The harvested epithelia were minced as finely as possible, and incubated on a type I collagen coated culture dish (BD BioCoat, Franklin Lakes, NJ, USA) for primary explant culture in KCM. After 2 weeks of primary culture, the proliferated cells were trypsinized, harvested, and seeded on a temperature-responsive cell culture insert (CellSeed, Tokyo, Japan) at a density of 5 × 104 cells/cm2. After a 10-day subculture on a temperature-responsive cell culture insert in KCM, harvesting of subcultured cells was attempted from the culture insert by lowering the culture temperature from 37 to 20 °C for 30 min.

Validation of culture sterility

Contamination of human nasal mucosal epithelial cell sheets was evaluated by using mycoplasma test and sterility test, using the culture supernatants. These tests were conducted using culture medium supernatant from the primary explant culture. Furthermore, bacterial and viral endotoxin tests were performed using cell culture supernatant. These tests were conducted at an independent clinical laboratory (SRL, Inc., Tokyo, Japan), according to the criteria and methods of the Japanese Pharmacopoeia guidelines [10].

Evaluation of cell sheet purity

To count the number of cells in a human nasal mucosal epithelial cell sheet, the latter was treated with 0.25% trypsin–0.1% EDTA (Invitrogen, Grand Island, N.Y., USA) for 20 min at 37 °C and filtered through a 40-μm cell strainer. Trypsinized cells were subsequently recovered and counted under a microscope. The cells were stained with trypan blue to evaluate the survival rate of the cells in the cell sheet. The living cells were counted under a phase contrast microscope with a 10× objective lens and cell survival rate calculated therefrom. To investigate cell purity in a manufactured nasal mucosal epithelial cell sheet, cytokeratin-positive cells were measured using a flow cytometer (EPICS XL EXPO32, Beckman Coulter, Palo Alto, CA, USA). Suspended nasal mucosal epithelial cells were fixed and permeabilized using a BD Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer's protocol; they were treated with FITC-conjugated anti-pan cytokeratin antibody (Progen, Heidelberg, Germany), and washed with BD Perm/Wash buffer (BD Biosciences) along with the BD Cytofix/Cytoperm Kit, following the manufacturer's protocol, before conducting flow cytometry analysis.

For histological cross-sectional analysis, the manufactured cell sheets were fixed with 10% neutral buffered formalin, processed into 3-μm-thick paraffin-embedded sections, and stained with hematoxylin and eosin (HE).

Soft-agar colony-formation assay

To investigate the presence of tumorigenic features in cultured human nasal mucosal epithelial cells, the ability of anchorage-independent growth was examined using soft-agar colony-formation assay. Anchorage-independent growth is one of the phenotypic changes related to malignant transformation [11,12]. The cells obtained by trypsinization of manufactured human nasal mucosal epithelial cell sheets were seeded at a density of 5 × 104 cells/cm2 in a 6-well plate filled with KCM containing 0.5% agar. The cells were cultured in a soft-agar medium for 15 days. HeLa cells were used as positive control, whereas fibroblasts (WI-38) were used as negative control. Both the cells were obtained from the Health Science Research Resources Bank (Osaka, Japan). After 15 days of culture, the number of colonies was counted. We verified whether the cells had characteristics of anchorage-independent growth based on the presence or absence of colony formation.

Results

Using nasal endoscopy, the inferior nasal concha mucosa was appropriately excised for preparing nasal mucosal epithelial cells from all four volunteer donors. The excised parts of inferior nasal concha were found not to re-bleed, and epithelized promptly in all four volunteer donors.

Manufacture of human nasal mucosal epithelial cells

In explant culture, epithelial cells were found to grow and move out from the piece of explant tissue after 7 days of culture (Fig. 3A). Proliferated epithelial cells with typical morphological features, such as polygonal cobblestone-shape, were observed from the periphery of the tissue fragments placed in the culture dishes (Fig. 3B). Following explant culture, the cells in the temperature-responsive cell culture inserts exhibited stable growth. The cells maintained their polygonal cobblestone shape even after two passages (Fig. 3C and D).

Fig. 3

Cultivation of human nasal mucosal epithelial cells. (A) Spread of nasal mucosal epithelial cells from the explant fragment and quick proliferation were observed after 7 days of culture. (B) The cells that spread from the explants were found to be of polygonal cobblestone shape with morphology typical of epithelial cells. (C) The cells cultured on temperature-responsive cell culture insert were observed to have a steady growth while retaining the cobblestone morphology even after two passages. (D) After a 12-day subculture, all cultured cells reached a confluent state and could be harvested as a single contiguous sheet by lowering the culture temperature from 37 °C to 20 °C.

Evaluation of human nasal mucosal epithelial cell sheets

After a 10-day subculture on the temperature-responsive cell culture inserts, all cells were successfully harvested as contiguous transplantable cell sheets (from the cell culture inserts) by lowering the incubation temperature from 37 °C to 20 °C for 30 min (Fig. 4A). All manufactured cell sheets exhibiting a stratified structure were histologically confirmed to compose of epithelial cells (Fig. 4B). The results indicated that the cultured human nasal mucosal epithelial cells could stratify on temperature-responsive cell culture inserts and cell sheets could be manufactured therefrom.

Fig. 4

Manufactured human nasal mucosal epithelial cell sheets. (A) Human nasal mucosal epithelial cells cultured on temperature-responsive cell culture insert were harvested as contiguous transplantable cell sheets. Bars indicate 5 mm. (B) All manufactured cell sheets were confirmed to be stratified structure composed of epithelial cells. Bars indicate 25 μm. (C) Measurement of cytokeratin-positive cells in a cell sheet using flow cytometry.

To verify sterility of human nasal mucosal epithelial cell sheets for clinical use, presence of bacterial, fungal, or virus contamination in the culture supernatants was checked.

In all samples, the results showed an absence of contamination by mycoplasma, viruses (HBV, HCV, HIV, and HTLV-1), aerobic bacteria, anaerobic bacteria, or lues venerea. In addition, endotoxin concentration was lower than 4.0 EU/mL in all samples (Table 1).

Table 1

Results of cell sheet sterility.

Test items	Volunteer number
	0001	0002	0003	0004
Mycoplasma	Negative	Negative	Negative	Negative
Endotoxin (EU/ml)	0.54	0.55	3.03	2.21
Bacteria and fungi
Aerobic bacteria	Negative	Negative	Negative	Negative
Anaerobic bacteria	Negative	Negative	Negative	Negative
Lues venerea	Negative	Negative	Negative	Negative
Fungi	Negative	Negative	Negative	Negative
Virus
HBV	Negative	Negative	Negative	Negative
HCV	Negative	Negative	Negative	Negative
HIV-1	Negative	Negative	Negative	Negative
HTLV-1	Negative	Negative	Negative	Negative

Characteristics of the manufactured cell sheets

The results of manufactured cell sheets are shown in Table 2. Three cell sheets were manufactured from one volunteer donor (number 0003) and six from the other donors. In all donors, the culture duration for fabricating cell sheets was 26 days. The cultured cells from all volunteer donors were successfully harvested as transplantable cell sheets from temperature-responsive cell culture inserts. All manufactured nasal mucosal epithelial cell sheets from donors (numbers 0001, 0002, 0003, and 0004) contained over 3.0 × 105 cells (7.5, 3.7, 3.0, and 5.1 × 105, respectively), and were able to maintain a cell survival rate of over 88% (93.6%, 92.0%, 89.3%, and 88.8% respectively). Furthermore, the number of cytokeratin-positive cells was determined in each cell sheet to confirm cell purity (Fig. 4C). All manufactured cell sheets from volunteer donors (numbers 0001, 0002, 0003, and 0004) contained over 89% (99.2%, 99.4%, 91.3%, and 89.2%, respectively).

Table 2

Characteristics of manufactured cell sheets.

	Volunteer number
	0001	0002	0003	0004
Age years/Sex	21/M	33/F	61/M	42/M
Number of sheet	6	6	3	6
Culture term days	26	26	26	26
Harvest test	Harvested	Harvested	Harvested	Harvested
Cell number (cells/sheet)	7.5 × 105	3.7 × 105	3.0 × 105	5.1 × 105
Cell survival rate (average %)	93.6%	92.0%	89.3%	88.8%
PCK-positive cells (%)	99.2%	99.4%	91.3%	89.2%

To verify the safety of cultured nasal mucosal epithelial cells for clinical use, the possibility of their phenotypic changes related to malignant transformation was examined. Human nasal mucosal epithelial cells, constituting a cell sheet, were cultured in a soft-agar medium to examine the possibility of anchorage-independent growth by the presence or absence of colony formation. HeLa cells, as the positive control, formed many colonies of several cells each, whereas fibroblasts and human nasal mucosal epithelial cells did not form colonies (Fig. 5).

Fig. 5

Results of soft-agar colony-formation assay. To ascertain the tumorigenic potential of human mucosal nasal epithelial cells, the obtained human nasal mucosal epithelial cells were seeded in keratinocyte culture medium (KCM) containing 0.5% agar. Human fibroblasts and human nasal mucosal epithelial cells formed no colonies, whereas HeLa cells formed many after 15 days culture.

Discussion

Clinical application of autologous nasal mucosal epithelial cell sheet transplantation is expected to potentially improve surgical treatment of intractable otitis media [2]. The current study aimed to develop nasal mucosal epithelial cell sheets as a product that can be applied clinically in near future. Elucidation of the possibility of clinical application of human nasal mucosal epithelial cell sheets would be of great importance before the start of a clinical study [13,14]. Here, we have established a system for evaluating the safety and validity of human nasal mucosal epithelial cell sheets.

When considering clinical cell transplantation, it is important to have a minimally invasive site for the collection of cell source. In this respect, the nasal mucosa, which can safely supply enough tissue with minimal invasion, was easily harvested without inflicting pain, and served as an excellent cell source.

The excised parts of the nasal mucosa were confirmed to not re-bleed and to epithelize promptly in all four volunteer donors.

The results of validation of culture sterility indicated the culture environment for manufacturing cell sheet to be compliant, and all procedures were conducted aseptically in the CPF. Analysis of flow cytometry results showed the percentage of cytokeratin-positive cells in the manufactured cell sheets to be higher than 89%. This supported our hypothesis that the manufactured cell sheets have characteristics of epithelial cells. Collectively, the results assured both safety and validity of manufactured human nasal mucosal epithelial cell sheets for clinical use.

The SOP was prepared to fabricate human nasal mucosal epithelial cell sheets for clinical transplantation into middle ear during otologic surgery. This SOP included the procedures of cultivation and manufacture of cell sheets in the CPF, and included methods of validation of sterility. We successfully manufactured cell sheets aseptically in the CPF. The purity of the manufactured cell sheets was confirmed by flow cytometry. All manufactured cell sheets were histologically confirmed to consist of epithelial cells. These results showed that the prepared SOP ensured stable manufacture and supply of cell sheets for clinical use. The number of cell sheets required for transplantation in middle ear surgery depends on the disease state, but at least three cell sheets are required, including one cell sheet for quality testing before transplantation. In this preclinical study, we were able to manufacture more than three cell sheets in each case, assuring that the protocol is feasible for clinical transplantation.

We confirmed the anchorage-dependent growth of cultured human nasal mucosal epithelial cells in the cell sheet using the soft-agar colony-formation assay. Malignant transformed cells change their phenotype to anchorage-independent growth [11,12]. The cultured human nasal mucosal epithelial cells were unable to grow in soft agar, therefore, indicating that the manufactured human nasal mucosal epithelial cells have no tumorigenic characteristic. This confirmed that there was no possibility of tumorigenesis in vivo after the transplantation of nasal mucosal epithelial cell sheets.

In this preclinical study, epithelial cells constituted the major population of cells in the cell sheets, as assessed using flow cytometry. However, the changes in the transplanted cell sheet at the transplantation site and the mechanism of its functioning is still unclear and needs further elucidation. It is difficult to predict the fate of the transplanted cell sheets, but we believe that it will depend on the individual treatment and the environment of the transplantation site. In middle ear surgery, cell sheet transplantation enables regeneration of the middle ear mucosa, as seen from clinical studies. In animal experiments, cells with epithelial-like structures are observed on the bone surface at the transplantation site, 8 weeks after the cell sheet transplantation, suggesting that these may be the transplanted cells [1]. However, cell sheets also exhibit paracrine functions [[15], [16], [17]]. Therefore, it is possible that the nasal mucosal epithelial cell sheets amplified the paracrine effect and regenerated the damaged residual middle ear mucosa. Considering these factors, we speculate a combination effect: the direct contribution of functioning tissue for the regeneration of middle ear mucosa and paracrine effects emanating from the cell sheet. Elucidating the fate of transplanted cells is very important for understanding the underlying mechanism of the effects of cell sheet therapy and therefore, is a critical question to be addressed in future studies.

This is beneficial not only in case of intractable otitis media, but also in other conditions like otolaryngology-head and neck surgery, any condition requiring early postoperative mucosal regeneration, such as sinusitis, pharyngeal and laryngeal stenosis, and tracheal stenosis. It is effective in inhibiting bone hyperplasia due to poor postoperative mucosal regeneration [1]; therefore, the human nasal mucosal epithelial cell sheets may be a good treatment tool for intractable frontal sinusitis caused by postoperative nasal mucosal defect and for mucosal regeneration suppression on the exposed bone surface. The nasal mucosa is the same mucosa as in pharynx, larynx, and trachea. The nasal mucosal epithelial cell sheets may therefore, be potent for reliable clinical application in various mucosal regeneration and tissue-reconstruction, as in pharynx, larynx, and trachea, in future. This study presents the possibility of a new treatment method for otorhinolaryngology. In Japan, laws related to regenerative medicine, such as the Act to Ensure the Safety of Regenerative Medicine, have been enacted. Based on these laws, Japan is expected to become a global hub of regenerative medicine, because of its position as a country advanced in regenerative medicine. The clinical application of cell sheet technology for regenerative medicine using is established for the cornea, heart, esophagus, and cartilage [[6], [7], [8], [9]]. However, regenerative medicine in the field of otorhinolaryngology is lagging. This preclinical study could contribute to the development of novel cell therapies for regenerative medicine, and we hope to revitalize research on regenerative medicine in the field of otorhinolaryngology.

Conclusions

Preclinical studies, such as this, are necessary for establishing safety in clinical research. Results of the current study suggested that cell sheets manufactured in the CPF are safe, with appreciable efficacy regarding mucosal regeneration and safety after transplantation in clinical settings. This study enabled the successful manufacture and clinical delivery of cell sheets. The findings could contribute significantly to the development of novel cell therapies in regenerative medicine.

Declaration of competing interest

Dr. Masayuki Yamato is an equity holder of CellSeed Inc., and Tokyo Women's Medical University currently receives research funding from CellSeed, Inc. Dr. Yamato is also an advisor of commercial efforts, Helios and NIPPI (Japan).