AdMSC sheets (PAL-222)

PAL-222 was manufactured and provided by PharmaBio Corporation using a recently developed cell incubation method [20, 21].

In vitro experiments to evaluate the PAL-222 propertiesViability of the cells obtained from PAL-222

PAL-222 was rinsed three times with phosphate buffered saline (PBS) in a 24-well plate. We added 200 µL of PBS to the well, detached PAL-222 using a micropipette (20–200 µL), and collected the sheets in a 1.5-mL tube. Collagenase (100 µL) at a concentration of 0.9 IU/mL was added to each tube, and the mixture was incubated at 37 °C for 15 min. Trypsin 0.25% (100 µL) was added to each tube and incubated at 37 °C for 5 min. The sheets were dissociated into single cells by pipetting with a micropipette (20–200 µL). The cell viability was assessed using the AO/PI Viability Assay Kit (CD-AO/PI-1.5, DeNovix, Inc., Wilmington, DE, USA) and the CellDrop FL instrument (DeNovix).

Cell surface antigens determined by flow cytometry

The expression levels of CD90, CD73, CD105, and CD44 in MSCs are considered essential markers for MSC confirmation [22]. Cell markers were analyzed as previously reported [23]. Dead cells in the single cell suspension were identified using Fixable Viability Stain 780 (FVS780, BD Biosciences, San Jose, CA, USA). The cells then were stained using the human MSC analysis kit (BD Biosciences, 562245). Negative markers associated with hematopoietic or immune cells (CD34, CD11b, CD19, CD45 and HLA-DR) were detected with the negative MSC cocktail in the kit. Stained cells were analyzed using the BD FACSLyric flow cytometer (BD Biosciences) (n = 9). Isotype controls were used in all analyses.

Secretion of VEGF and PEDF for 24 h by cultured PAL-222

We evaluated the VEGF and PEDF secretory ability of PAL-222, both of which are associated closely with retinoprotective effects. PAL-222 was stored for 96 h and then placed in an incubator (37 °C, 5% CO2). After a 24-h incubation, the culture supernatant was collected and analyzed. VEGF was measured using the Quantikine ELISA Human VEGF Immunoassay (Cat. No. DVE00, R&D Systems, Newark, DE, USA). PEDF was measured using Human Serpin F1/PEDF (Cat. No. DY1177-05, R&D Systems) and DuoSet Ancillary Reagent Kit 2 (Cat. No. DY008, R&D Systems). The samples for VEGF measurement were diluted to 1:40 using a Calibrator Diluent RD5K. The samples for PEDF measurement were diluted to 1:40 with the reagent diluent. We quantified the secreted VEGF and PEDF levels using a multiplate reader (PerkinElmer Japan, Oita, Japan, EnVision, Cat. No. 2104) (n = 9).

Immunohistologic examination for cellular and tissue antigens

We chose PAL-222, anticipating that it would have a structure similar to Bruch’s membrane and functions close to those expressed by Bruch’s membrane. An immunohistologic analysis was conducted to examine if PAL-222 exhibited structure and function similar to Bruch’s membrane. Type I collagen (an index of strength), type IV collagen (an index of a basement membrane), and elastin (an index of elasticity) were selected as markers that characterize the functions of the sheet, and their expression was checked on PAL-222. After fixation of PAL-222 with 4% paraformaldehyde (Cat. No. 09154-56, Nacalai Tesque, Inc., Kyoto, Japan), blocking with 3% BSA/PBS/0.5% TritonX-100 was conducted for 60 min at room temperature. After overnight incubation at 4 °C with each of the 1:100 diluted primary antibodies to type I collagen (Cat. No. ab34710, Abcam, Waltham, MA, USA), type IV collagen (Cat. No. ab6586, Abcam), and elastin (Cat. No. ab23747, Abcam), the samples were exposed to 1:1000 diluted secondary antibody labeled with Alexa Fluor 488 (Abcam) for 60 min at room temperature. No primary antibody was applied to control samples. For counterstaining, DAPI solution (Cat. No. 340–07971, Dojindo, Rockville, MD, USA) was used. After inclusion, the samples were observed under a Lionheart FX fluorescence microscope (Bio Tek Instruments, Winooski, VT, USA). Images were analysed using FIJI software [24]. Established threshold value was applied to the images to quantify the expression of type I collagen, type IV collagen, and elastin. Expression area (%) was calculated as the ratio of the expression area to the total area.

Transwell in vitro migration assay

A transwell in vitro migration assay was performed to assess the durability and similarity of PAL-222 to RPE using fetal bovine serum (FBS) as a chemoattractant for cells. For adMSC suspension, cells were treated in 800 µL of α-MEM/GlutaMax (GIBCO, Grand Island, NY, USA) with or without chemoattractants (40% or 20% FBS) per well in a 24-well plate. Cell suspensions (1.0 × 105 live adMSCs) were seeded in inserts with a pore size of 8 μm. Twenty-four hours later, the cells in the inserts were removed, and cell count was evaluated using 0.4% Crystal Violet Stain (Sigma-Aldrich) and Cell Counting Kit-8 (CCK-8, Dojindo Laboratories Inc.) (n = 3 in each group). PAL-222 in 8-µm-pore transwell inserts was provided by PharmaBio Corporation. We prepared another 24-well plate with 800 µL of α-MEM/GlutaMax with or without chemoattractants (40% or 20% FBS). The medium in the inserts of the cell sheets was removed, and 200 µL of α-MEM/GlutaMax was added to each well. The inserts were transferred to the other 24-well plate. Twenty-four hours later, the cells in the inserts were removed, and the cell count was assessed using 0.4% Crystal Violet Stain and CCK-8 (n = 3 in each group). The procedure involved applying 0.4% Crystal Violet Stain and incubating for 10 min. Subsequently, Crystal Violet dissolved in a 33% acetic acid solution, and the eluate was added to a 96-well plate. Absorbance was measured at 620 nm using a plate reader. On the other hand, CCK-8 was added to the wells and incubated for 2 h. Absorbance within the range of 450 nm to 620 nm was measured using a plate reader.

In vitro experiments to evaluate the tumorigenicity of PAL-222

To assess in vitro tumorigenicity, we performed a chromosomal aberration test and a colony formation assay with a soft agar medium using the decomposed cells obtained from overcultured adMSC sheets as preclinical safety tests.

Chromosomal aberration test

To assess the functional abnormalities, transformations, and mutations of single cells obtained by enzymatic treatment of PAL-222 produced using cells subcultured within and beyond the specified culture period, chromosome analysis was performed to examine abnormalities in chromosome composition. The adMSCs obtained from PAL-222 were cultured beyond the specified culture period in the test cell group. The population doubling level (PDL) was 15. On the other hand, the adMSCs obtained from PAL-222 were cultured within the specified period in the control cell group. The PDL was 8.5. After cell expansion in each group, metaphase cells were collected for chromosomal analysis. We analyzed 50 cells to evaluate the number of chromosomes, sex chromosome configuration, G-banding pattern (20 cells), and mode analysis (30 cells). We analyzed samples stained by G-banding, which performs differential staining using the trypsin method. We determined a karyotype that conforms to the International System for Human Cytogenetic Nomenclature, 2020 [25].

Colony formation assay with a soft agar medium

The soft agar colony formation test detects anchorage-independent cell proliferation and is used to detect malignant transformed cells that are mixed in or generated during the manufacturing process of cell processed products. Single cells were obtained by enzymatic treatment of PAL-222 from cells subcultured beyond the specified culture period and were assessed for colony formation, indicating anchorage-independent growth, after three weeks of culture in soft agar medium. The adMSCs obtained from PAL-222 were cultured with PDL 15 beyond the specified culture period (n = 4). We selected HeLa cells (a cell strain derived from human cervical cancer) as positive control (n = 4) and MRC-5 cells (normal human diploid fibroblasts derived from fetal lung) as negative control (n = 4). Cells were collected in the soft agar medium, cultured for 3 weeks. Colonies were observed using a microscope, and the numbers, lengths, and widths of the colonies were measured. Cell aggregates that were not observed at the start of the culture and exhibited a long axis of 100 μm or more were identified as representing anchorage-independent cell proliferation. Consequently, they were classified as colonies. The test was considered successful if one or more colonies were formed in the positive control group, and no colonies with a long diameter of 100 μm or more were formed in the negative control group after three weeks of culture.

In vivo nonclinical pharmacologic study to assess the safety and retinoprotective effects of PAL-222 implantation in RCS rats

The work has been reported in line with the ARRIVE guidelines 2.0. The safety and retinoprotective effects of transplanted PAL-222 were evaluated in Royal College of Surgeons (RCS Jcl-rdy/rdy) rats, an animal model with congenital retinal degeneration, in Safety Research Laboratory of Shin Nippon Biomedical Laboratories, Ltd. (2438 Miyanouracho, Kagoshima City, Kagoshima Prefecture). Three-week-old male RCS rats with inherited retinal degeneration were purchased from Clea Japan, Inc. (Fujinomiya City, Shizuoka, Japan). Since it is considered that there is no difference in efficacy between males and females, only male rats were used in the study. The male RCS rats began acclimatization on the day of arrival. The rats were raised in a room under a 12-h light/dark cycle at room temperature and had free access to a solid diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) and water until group allocation. The day of group allocation was counted as day − 1, the day of transplantation as day 1, and the week of transplantation as week 1. Groups were assigned by stratified randomization according to weight using a safety test system (Mitsui Toxicological Data Processing System, MiTOX, Mitsui E&S Systems Research Inc., Chiba, Japan) so that there was no difference in weight between groups. Only the employees of a third-party company (Safety Research Laboratory of Shin Nippon Biomedical Laboratories, Ltd.) were aware of the group allocation during the experiments including the allocation, the conduct of the experiments, the outcome assessment, and the data analysis. The authors remained blinded to the group assignment throughout the study to minimize potential bias and ensure the integrity of the study results. For PAL-222 group, ten RCS rats were considered sufficient to confirm the safety and efficacy of PAL-222. For the sham group, five RCS rats were considered sufficient for statistical analysis. Based on body weight, 15 RCS rats were randomly divided into the sham group (n = 5) and PAL-222 group (n = 10) for efficacy evaluation for 3 weeks. After the division into groups, the rats were allowed free access to immunosuppressant (0.21 mg/mL cyclosporine solution, Novartis Pharmaceuticals Corporation, Tokyo, Japan) supplied as drinking water from the water feeder throughout the observation period. Toys for rats were always available. For each experimental group, there were no animals, experimental units, or data points that were not included in the analysis. Confounders were not controlled in this study. For PAL-222 transplantation procedures, a conjunctival incision was made 1.0 mm posterior to the limbus on the temporal side of the right eyes and a 2.0-mm scleral incision was created at the limbus under inhalational anesthesia with 1.0–4.0% isoflurane (Isoflurane Solution for Inhalational Anesthesia Pfizer; Mylan Inc., Canonsburg, PA, USA). A 30-gauge needle was inserted at an approximately 15-degree angle that penetrated the choroid and RPE to create an artificial retinal detachment with a diameter of approximately 3 mm using VISCOAT 0.5 Ophthalmic Viscoelastic Substance (Alcon Japan Ltd.). Subsequently, one piece of PAL-222 was transplanted into the subretinal lower space of the detached area only in the PAL-222 group. An antibiotic (Clavit Ophthalmic Solution 0.5%, Santen Pharmaceutical Co., Ltd., Osaka, Japan) was applied to both eyes for 3 days after the procedure. After transplantation of the sheet, the safety and retinoprotective activity were evaluated by conducting the following assessments for 3 weeks. Observation and inspection items include general condition, body weight, ophthalmologic examinations, necropsy, and histopathologic examination. The general conditions were monitored daily. Body weight was measured using an electronic scale (GX-4000, A&D Company, Limited) on days − 1, 5, 6, 8, 12, 15, 19 and 22. Weight gain at each weighing was also determined. Ophthalmologic examinations were performed on the 15th day. A portable slit lamp for animals (SL-17, Kowa Company, Ltd., Aichi, Japan) was used for gross ophthalmologic examination and pupillary light reflex testing. After instillation of mydriatic solution (tropicamide and phenylephrine hydrochloride, Santen Pharmaceutical Co., Ltd.), the anterior segment and ocular media were examined with the portable slit lamp (SL-17). Fundus examination was performed with a binocular indirect ophthalmoscope (IO-α Small Pupil, Neitz Instruments Co., Ltd., Tokyo, Japan). Since no abnormalities were found in the fundus, no fundus photographs were taken. The rats were euthanized by exsanguination under isoflurane inhalation anesthesia (2.0–4.0%) on the day after the end of the observation period, and the external appearance, internal organs, and tissues were observed macroscopically at necropsy for pathological examination. Rat eyes and optic nerves were fixed in a mixture of 3% glutaraldehyde and 2.5% formalin. The eyes and optic nerves were embedded in paraffin, sectioned, and stained with hematoxylin-eosin (HE) stain for histopathologic examination. The thickness of outer nuclear layer (ONL), which corresponds to the photoreceptor nuclei, in the sham and PAL-222 groups was measured using Aperio ImageScope 12.4.6 (Leica Biosystems, Nussloch, Germany). In the statistical analysis, the weight of each group was tested for equal variance between the sham and PAL-222 groups using F-test, Student’s t-test for equal variance, and Aspin-Welch test for unequal variance. The statistical analysis was performed using the safety test system (MiTOX), with a two-sided significance level of 5%. The histopathologic data on the ONL thickness were compared between the sham and PAL-222 groups using Student’s t-test with Stata/BE 18.0 for Windows (StataCorp LLC, USA). P values < 0.05 were considered statistically significant. General conditions, ophthalmologic examinations, and necropsy data were not statistically examined.