JP7844055B2 - Multilayer cell structure and method for producing the same - Google Patents

Description

This invention relates to a multilayer cell structure and a method for producing the same.

In current regenerative medicine research and clinical practice, temperature-responsive culture dishes are widely used. Cells are cultured in these temperature-responsive dishes, and when the temperature is lowered from 37°C to 20°C, the cells can be harvested in a sheet. However, the harvested cell sheets are fragile and single-layered. For transplantation, it is necessary to stack the sheets one by one using a cell sheet support to create a multi-layered sheet, which is time-consuming and labor-intensive.

One of the representative somatic stem cells used in regenerative medicine is mesenchymal stem cells (hereinafter sometimes referred to as "MSCs"). Examples of MSCs include stem cells derived from bone marrow, umbilical cord, teeth, etc.

Generally, the culture medium used for culturing MSCs requires the addition of 10-20% fetal bovine serum (FBS). However, because FBS is animal-derived, there are issues such as immune reactions due to heterologous antigens, infection by prions and viruses, and lot-to-lot variations in FBS components. These present many challenges that need to be addressed to provide safe and stable regenerative medicine.

Therefore, in recent years, there has been a demand for culture media that do not use FBS (hereinafter sometimes referred to as "FBS-free" or "serum-free"), and serum-free culture media that do not have problems with immunity, infection, or lot-to-lot variation have been developed. Examples of such serum-free culture media include serum-free culture media (XFM, manufactured by Irvine Scientific) manufactured under current pharmaceutical good manufacturing practice (cGMP).

To date, it has been reported that dental pulp stem cells cultured in XFM (hereinafter sometimes referred to as "XFM cells") yield approximately twice the number of cells by day 12 of culture compared to dental pulp stem cells cultured in conventional 15% FBS-containing culture medium (hereinafter sometimes referred to as "SCM cells") (see Non-Patent Literature 1). Furthermore, Non-Patent Literature 1 reports that, in the confluent state (where cells fill the petri dish) after 8 days of culture, SCM cells maintain a monolayer, while XFM cells autonomously multilayer (hereinafter sometimes referred to as "self-multilayering") form a cell sheet-like structure. It also reports that the number of XFM cells decreased on 14 days of culture, and that when cell death was analyzed using TUNEL (Terminal deoxynucleotydyl transfer-mediated deoxyuridine triphosphosphate nick-end labeling) staining, a very large number of TUNEL-positive cells were observed in XFM cells.
Therefore, while culturing with XFM can increase the cell count by about twice as much as with conventional methods, there was a problem in that self-multilayered cells underwent cell death, resulting in a decrease in the cell count.

As a follow-up to Non-Patent Literature 1, in order to solve the problem of cell death occurring due to self-multilayering, attention was focused on the coating of culture equipment (Petri dishes and plates), and it was reported that type I collagen is the optimal coating substrate (see Non-Patent Literature 2). Non-Patent Literature 2 reports that dental pulp stem cells cultured in culture dishes coated with collagen (hereinafter sometimes referred to as "COL-XFM cells") continued to proliferate even more sustainably than XFM cells, and the number of cells did not decrease until day 20 of culture, ultimately succeeding in forming a self-multilayered cell sheet that reached approximately twice the number of cells. Furthermore, when this self-multilayered cell sheet on day 20 of culture was sectioned and stained with TUNEL, it was confirmed that there were fewer TUNEL-positive cells than XFM cells. In addition, in the analysis of mitochondrial membrane potential by flow cytometry using JC-1, it was reported that there was no significant change in the membrane potential of COL-XFM cells from day 10 to day 20 of culture, while a significantly larger amount of JC-1 monomer produced by the decrease in membrane potential was observed in XFM cells.
The results above indicate that COL-XFM cells reach a plateau in cell number and produce an extracellular matrix (hereinafter sometimes referred to as "ECM"), forming a mature, self-multilayered cell sheet on day 20 of culture. However, a considerable number of cells within the sheet exhibit TUNEL positivity and a certain degree of membrane potential reduction.

Mochizuki et al. , Establishment of xenogenic serum-free culture methods for handling human dental pulp stem cells using clinically oriented in-vitro and in-vivo conditions, Stem Cell Research & Therapy (2018) 9:25 Mochizuki et al. , Type I collagen facilitates safe and reliable expansion of human dental pulp stem cells in xenogenic serum-free Culture, Stem Cell Research & Therapy (2020) 11:267

The present invention aims to solve the aforementioned conventional problems and achieve the following objectives. Specifically, the present invention aims to provide a method for producing a multilayer cell structure and a multilayer cell structure that can shorten the culture period required for the production of the multilayer cell structure, suppress cell death in the multilayer cell structure, and produce multilayer cell structures of various shapes.

The present inventors conducted diligent studies to achieve the above objectives and discovered that when culturing mesenchymal stem cells derived from dental pulp using serum-free medium and culture equipment coated with type I collagen or its fragments, by setting the seeding rate of the dental pulp-derived mesenchymal stem cells to more than 0.5 × ^10⁴ cells/ ^cm² and less than 2 × ^10⁴ cells/ ^cm² , the culture period required for the production of multilayer cell structures can be shortened, cell death in the multilayer cell structures can be suppressed, and furthermore, multilayer cell structures of various shapes can be produced. Based on these findings, the present invention was completed.

The present invention is based on the inventors' aforementioned findings, and the means for solving the aforementioned problems are as follows:
<1> This includes culturing mesenchymal stem cells derived from dental pulp using serum-free medium and culture equipment coated with type I collagen or its fragments,
The method for producing a multilayer cell structure is characterized in that the seeding number of mesenchymal stem cells derived from dental pulp is greater than 0.5 × ^10⁴ cells/ ^cm² and less than 2 × ^10⁴ cells/ ^cm² .
<2> The serum-free medium is a method for producing the multilayer cell structure described in <1> above, which does not contain components from a different animal species.
<3> A method for producing a multilayer cell structure according to any one of <1> to <2>, wherein the shape of the multilayer cell structure is a sheet, a rod, or a disc.
<4> A method for producing the multilayer cell structure described in <3>, wherein the shape of the multilayer cell structure is sheet-like and has a thickness of 5 μm to 40 μm.
<5> A method for producing a multilayer cell structure according to any one of <1> to <4>, wherein the proportion of dead cells in the multilayer cell structure is 5% or less.
<6> A method for producing the multilayer cell structure according to any one of <1> to <5>, wherein the multilayer cell structure expresses type I collagen, type IV collagen, and type VII collagen.
<7> A multilayer cell structure of mesenchymal stem cells derived from dental pulp,
The multilayer cell structure is characterized in that the proportion of dead cells in the multilayer cell structure is 5% or less.
<8> The multilayer cell structure is the multilayer cell structure described in <7> that expresses type I collagen, type IV collagen, and type VII collagen.
<9> The multilayer cell structure according to any one of <7> to <8>, wherein the shape of the multilayer cell structure is one of a sheet, a rod, or a disc.
<10> The multilayer cell structure described in <9>, wherein the shape of the multilayer cell structure is sheet-like and has a thickness of 5 μm to 40 μm.

According to the present invention, it is possible to solve the aforementioned conventional problems and achieve the aforementioned objectives, shorten the culture period required for the production of multilayer cell structures, suppress cell death in multilayer cell structures, and provide a method for producing multilayer cell structures and multilayer cell structures that can be made into multilayer cell structures of various shapes.

Figure 1 is a graph showing the results of the growth curve evaluation in Test Example 1. Figure 2 is a phase-contrast micrograph of the growth curve evaluation for Test Example 1 on day 10 of culture. Figure 3 is a graph showing the results of the growth curve evaluation in Test Example 2. Figure 4 is a phase-contrast micrograph of the growth curve evaluation in Test Example 2 on day 10 of culture. Figure 5 shows stereomicroscope images of cell sheets from test examples 2-1 and 2-2 on day 10 of culture. Figure 6 shows a stereomicroscope image of the cell sheet from Test Example 2-3 after 10 days of culture. Figure 7 shows the results of hematoxylin-eosin (HE) staining and Masson's trichrome (MT) staining of cells cultured on day 10 of Test Example 2. Figure 8 is an immunofluorescence staining diagram showing the results of TUNEL staining and DAPI staining of cells cultured on day 10 of Test Example 2. Figure 9 shows the results of immunofluorescence staining of various collagens in the autologous multilayer cell sheet of dental pulp stem cells cultured on day 10 of Test Example 2-1. Figure 10 shows the appearance of the autologous multilayered cell sheet of dental pulp stem cells after 10 days of culture in Test Example 3, when it has been rolled up. Figure 11 shows the appearance of the rolled-up state after 4 days of culture in Test Example 3. Figure 12 shows the results of hematoxylin-eosin (HE) staining of a cross-section of the rolled specimen from Test Example 3 after 4 days of culture. Figure 13 shows the results of immunofluorescence staining of various collagens in a cross-section of a cell tube on day 4 of culture after roll-up in Test Example 3. Figure 14 shows a phase-contrast microscope image of cells from Test Example 4 after one month of culture. Figure 15 shows a stereomicroscope image of cells from Test Example 4 after one month of culture (before being loaded with tweezers). Figure 16 shows a stereomicroscope image of cells from Test Example 4 after one month of culture (when held with tweezers). Figure 17 shows the results of hematoxylin-eosin (HE) staining of cells from Test Example 4 after one month of culture. Figure 18 shows the results of Masson's trichrome (MT) staining of cells from Test Example 4 after one month of culture. Figure 19 is an immunofluorescence staining diagram showing the results of TUNEL staining and DAPI staining of cells from Test Example 4 after one month of culture. Figure 20 shows the results of immunofluorescence staining of various collagens in dental pulp stem cells one month after culture in Test Example 4-1. Figure 21 is a graph showing the results of the growth curve evaluation in Test Example 5. Figure 22 is a phase-contrast micrograph of the growth curve evaluation for Test Example 5 on day 10 of culture. Figure 23 shows stereomicroscope images (when supported with tweezers) of autologous multilayered cell sheets of dental pulp stem cells from test examples 5-1 and 5-2 on day 10 of culture. Figure 24 shows the results of hematoxylin-eosin (HE) staining and Masson's trichrome (MT) staining of cells cultured on day 10 of Test Example 5. Figure 25 shows the results of immunofluorescence staining of various collagens in a cell sheet of dental pulp stem cells cultured 10 days before the end of Test Example 5-1. Figure 26 shows the results of immunofluorescence staining of various collagens in a cell sheet of dental pulp stem cells cultured 10 days after the start of the experiment in Example 5-2.

(Multilayer cell structure and method for producing the same)
The present invention provides a method for producing a multilayer cell structure, comprising at least a culture step and, if necessary, further other steps.
The multilayer cell structure of the present invention can be suitably manufactured by the method for manufacturing the multilayer cell structure of the present invention.
The following describes the method for producing the multilayer cell structure of the present invention, along with the multilayer cell structure itself.

<Culture process>
The aforementioned culture step involves culturing mesenchymal stem cells derived from dental pulp (hereinafter sometimes referred to as "dental pulp stem cells") using serum-free medium and culture equipment coated with type I collagen or its fragments.

<<Serum-free medium>>
As for the serum-free medium, there are no particular restrictions as long as it does not contain FBS, and any known mesenchymal stem cell proliferation medium can be appropriately selected depending on the purpose.
The serum-free medium may be a commercially available product.
There are no particular restrictions on the commercially available serum-free culture medium, and it can be appropriately selected depending on the purpose. Examples include PRIME-XV® MSC Expansion XSFM (manufactured by Irvine Scientific Corporation/Fujifilm Wako Pure Chemical Industries, Ltd.).

The serum-free culture medium may or may not contain components from other animal species, but it is preferable that it does not contain components from other animal species.

<<Culture equipment>>
The culture equipment is not particularly limited as long as it is coated with type I collagen or a fragment thereof, and known culture equipment can be appropriately selected according to the purpose.
There are no particular restrictions on the form of the culture equipment, and it can be appropriately selected according to the purpose. Examples include culture dishes (petri dishes), culture plates, etc.

The aforementioned type I collagen fragment refers to a fragment that has part of the amino acid sequence of type I collagen. The aforementioned type I collagen fragment may also be a fragment formed by linking together multiple overlapping portions of the amino acid sequence of type I collagen.
The aforementioned type I collagen fragments may be recombinant proteins (hereinafter sometimes referred to as "recombinant peptides") or they may be fragments obtained by degrading type I collagen.

There are no particular restrictions on the type I collagen or its fragments, and commercially available products can be used as appropriate. For example, a commercially available type I collagen fragment is cellnest Human Type I Collagen-like Recombinant Peptide (manufactured by Fujifilm Corporation).
The method and amount of applying the type I collagen or its fragments to the culture equipment are not particularly limited, as long as they do not impair the effects of the present invention. Known methods and application amounts can be appropriately selected according to the purpose, for example, the application method and application amount recommended by the manufacturer.

The culture equipment may be one that has type I collagen or a fragment thereof coated on it at the time of use, or it may be one that has already been coated.
The culture equipment may be commercially available.

<<Dental pulp stem cells>>
The dental pulp stem cells may be those prepared from teeth at the time of use, or pre-prepared dental pulp stem cells may be used.
There are no particular restrictions on the species of dental pulp stem cells, and they can be appropriately selected according to the purpose. Examples include humans, mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, cattle, pigs, and monkeys.

There are no particular limitations on the method for preparing the dental pulp stem cells, and known methods can be appropriately selected depending on the purpose. For example, in humans, methods include preparing them from extracted teeth of adults or from deciduous teeth during the mixed dentition stage (exchange stage).

There are no particular restrictions on the dental pulp stem cells used in the culture process described above; they can be appropriately selected according to the purpose. For example, dental pulp stem cells with 3 to 5 passages can be used.

<<Culture>>
The number of dental pulp stem cells seeded in the culture step is not particularly limited as long as it is greater than 0.5 × ^10⁴ cells/ ^cm² and less than 2 × ^10⁴ cells/ ^cm² , and can be appropriately selected according to the purpose. However, 0.75 × ^10⁴ cells/ ^cm² or more and less than 2 × ^10⁴ cells/ ^cm² is preferred, 0.75 × ^10⁴ cells/cm² or ^more and 1.5 × ^10⁴ cells/ ^cm² or less is more preferred, and greater than 0.75 × ^10⁴ cells/ ^cm² and 1.5 × ^10⁴ cells/ ^cm² or less is particularly preferred.

The temperature and carbon dioxide ( _CO₂ ) concentration in the culture are not particularly limited as long as they do not impair the effects of the present invention, and known conditions for culturing mesenchymal stem cells can be appropriately selected, for example, a temperature of 37°C and a _CO₂ concentration of 4.7%.

There are no particular restrictions on the culture period, and it can be appropriately selected depending on the shape of the desired multilayer cell structure. According to the method of the present invention, self-multilayered cell sheets can be produced in a shorter period than conventional methods.

Other conditions in the culture described above are not particularly limited as long as they do not impair the effects of the present invention, and known conditions for culturing mesenchymal stem cells can be appropriately selected.

<Other processes>
The aforementioned other steps are not particularly limited as long as they do not impair the effects of the present invention, and can be selected as appropriate. For example, these may include the step of preparing the dental pulp stem cells described above.

<Multilayer cell structure>
The multilayer cell structure of the present invention is a multilayer cell structure of mesenchymal stem cells derived from dental pulp. Preferably, the multilayer cell structure is self-multilayered (hereinafter sometimes referred to as "self-multilayered tissue").
In this specification, self-multilayering refers to the autonomous multilayering of cells, and it is preferable that the cells produce extracellular matrix (ECM).

There are no particular restrictions on the number of layers in the aforementioned multilayer cell structure, as long as there are two or more layers, and they can be appropriately selected according to the purpose.
There are no particular restrictions on the method for confirming whether the aforementioned multilayer cell structure is multilayered, and a suitable method can be selected depending on the purpose. Examples include pathological confirmation using hematoxylin-eosin (HE) staining or Masson's trichrome (MT) staining, and confirmation using a confocal laser microscope after fluorescently staining specific proteins inside and outside the cell. Furthermore, while a confluent monolayer structure on the culture medium is transparent, a multilayered cell structure causes the surface of the culture medium to become thicker and cloudy, so the approximate state of multilayering can also be grasped by visual inspection of the culture medium.

There are no particular restrictions on the shape of the multilayer cell structure, and it can be appropriately selected depending on the purpose. Examples include sheet-like (hereinafter sometimes referred to as "cell sheet"), rod-like (hereinafter sometimes referred to as "cell tube"), and disc-like (hereinafter sometimes referred to as "cell disc").

There are no particular restrictions on the thickness of the cell sheet, and it can be appropriately selected depending on the purpose. For example, it can be 5 μm to 40 μm, and preferably 10 μm to 35 μm. The thickness of the cell sheet refers to the average value of the thickness measured at 10 randomly selected locations on the cell sheet.
The cell sheet can be formed by performing the culture process described above for about 10 days.
There are no particular restrictions on the area of the cell sheet when viewed from above, and it can be appropriately selected depending on the purpose.

The aforementioned cell tube is rod-shaped. It can also be described as a solid, tubular structure.
The cell tube can be formed, for example, by rolling a cell sheet obtained by culturing in the culture process described above for about 10 days into a tube shape, and then culturing it for a further 4 days.
There are no particular restrictions on the thickness or length of the cell tubes, and they can be appropriately selected according to the purpose.
There are no particular restrictions on the method for confirming whether the cell tube is solid or not, and a suitable method can be selected depending on the purpose. Examples include hematoxylin-eosin (HE) staining.

There are no particular restrictions on the thickness of the cell disk, and it can be appropriately selected depending on the purpose. For example, it can be greater than 40 μm and less than or equal to 60 μm.
The cell disk can be formed by performing the culture process described above for about one month.
There are no particular restrictions on the area of the cell disk when viewed from above, and it can be appropriately selected depending on the purpose.

There are no particular restrictions on the mechanical strength of the cell sheet, cell tube, and cell disk, and they can be appropriately selected according to the purpose. For example, the cell sheet can have a compressive strength of about 0.02 N to 0.04 N, the cell tube can have a tensile strength of about 0.2 N to 0.5 N, and the cell disk can have a compressive strength of about 0.5 N to 1.0 N and a tensile strength of about 0.1 N to 0.5 N.

The aforementioned multilayer cell structure exhibits suppressed cell death.

There are no particular restrictions on the percentage of dead cells in the aforementioned multilayer cell structure, and it can be appropriately selected depending on the purpose, but it is preferably 5% or less, more preferably 3% or less, and particularly preferably 1% or less.
There are no particular limitations on the method for analyzing the proportion of dead cells in the aforementioned multilayer cell structure, and a suitable method can be selected depending on the purpose. For example, a method using the Live or Dead Cell Viability Assay Kit (manufactured by AAT Bioquest) can be used.

Furthermore, there are no particular restrictions on the method for confirming whether or not dead cells are present in the multilayer cell structure, and a suitable method can be selected depending on the purpose. For example, this can be done by TUNEL (Terminal deoxynucleotydyl transfer-mediated deoxyuridine triphosphosphate nick-end labeling) staining.
There are no particular restrictions on the method of TUNEL staining; for example, it can be performed using TUNEL staining manufactured by Roche Diagnostics.
Preferably, the aforementioned multilayer cell structure is one in which no TUNEL-positive cells are detected when stained with TUNEL.

The aforementioned multilayer cell structure preferably expresses type I collagen, type IV collagen, and type VII collagen.
There are no particular limitations on the method for confirming whether the aforementioned multilayer cell structure expresses type I collagen, type IV collagen, and type VII collagen, and a suitable method can be selected depending on the purpose. For example, this could be done by immunofluorescence staining using antibodies against each type of collagen.

There are no particular limitations on the use of the aforementioned multilayer cell structure, and it can be appropriately selected according to the purpose. For example, it can be suitably used as a material for regenerative medicine.
Specifically, the cell sheet can be suitably used as a regenerative medicine material for, for example, bone, periodontal tissue, teeth (dentin, etc.), skin (epidermis, dermis, etc.), corneal epithelium, cartilage, brain, heart, liver, pancreas, kidney, uterus, middle ear, thyroid gland, esophagus, lungs, etc.; the cell tube can be suitably used as a regenerative medicine material for, for example, nerves, muscles, blood vessels, teeth (dentin, etc.), tendons, ligaments, etc.; and the cell disc can be suitably used as a regenerative medicine material for, for example, teeth (dentin, etc.), skin (epidermis, dermis, etc.), corneal epithelium, temporomandibular joint disc, knee cartilage, bone, etc.

The present invention will be explained below with reference to test examples, but the present invention is not limited in any way to these test examples.

(Test example 1)
<Preparation of dental pulp stem cells>
Extracted wisdom teeth from adult humans were placed in a basal culture medium free of fetal bovine serum (FBS) [Dulbecco's modified Eagle's medium/Ham's nutrient mixture F12 (DMEM/F12), 100 μM glutamate, 0.1% MEM nonsensical amino acids, 50 U/mL penicillin, 50 μg/mL streptomycin, and 0.25 mg/mL fungizone (all from Thermo Fisher Scientific)], transported to the laboratory at 4°C, and pulp tissue was collected aseptically.

Dental pulp tissue was incubated for 1 hour at 37°C in a mixture of 3 mg/mL collagen type I (Merck KGaA) and 4 mg/mL discharge (Fujifilm Wako Pure Chemical Industries), and a suspension of dental pulp stem cells was prepared by passing the mixture through a cell strainer (φ70 μm; Greiner Bio One).

Following standard procedures, dental pulp stem cells were seeded in 60 mm culture dishes (manufactured by Thermo Fisher Scientific, hereafter referred to as culture dishes pre-coated with type I collagen) coated with type I collagen (Cellmatrix I; manufactured by Nitta Gelatin Co., Ltd.), and cultured at 37°C in a cGMP xeno-/serum-free culture medium (XFM) (PRIME-XV® MSC Expansion XSFM; manufactured by Irvine Scientific/Fujifilm Wako Pure Chemical Industries, Ltd.) under a 4.7% _CO2 environment. Once the cells had grown to 80% confluence, dental pulp stem cells were harvested using a mixture of 0.25% trypsin (Becton Dickinson) and 0.02% ethylenediaminetetraacetic acid (EDTA; Dojindo). These cells were then subcultured in 60 mm culture dishes at a cell density of 5 × ^10³ cells/ ^cm² (0.5 × ^10⁴ cells/ ^cm² ), and dental pulp stem cells at passage 3 to 5 were used in the following experiments.

<Evaluation of growth curves>
Dental pulp stem cells were seeded in 12-well culture dishes under the following four conditions and cultured for 12 days at 37°C in a 4.7% _CO2 environment using cGMP xeno-/serum-free culture medium (XFM) (PRIME-XV® MSC Expansion XSFM; manufactured by Irvine Scientific Co., Ltd./Fujifilm Wako Pure Chemical Industries, Ltd.). Cell counts were recorded every two days. The results are shown in Figure 1.
• Test Example 1-1: 0.5 × ^10⁴ cells/ ^cm² (indicated by "◆" in Figure 1)
• Test example 1-2: 1 × ^10⁴ cells/ ^cm² (indicated by "□" in Figure 1)
• Test example 1-3: 2 × ^10⁴ cells/ ^cm² (indicated by "▲" in Figure 1)
• Test example 1-4: 4 × ^10⁴ cells/ ^cm² (indicated by "○" in Figure 1)

As shown in Figure 1, cell proliferation was faster in test examples 1-2 to 1-4 than in test example 1-1. In test example 1-2, automultilayering was observed on day 10 of culture. On the other hand, in test examples 1-3 and 1-4, as can be seen from the large standard deviation (±SD), there was variability in cell proliferation, and automultilayering was not observed even on day 10 of culture.

In test examples 1-2 to 1-4, phase-contrast microscope images taken on day 10 of culture are shown in Figure 2.
In Test Examples 1-2, the culture dish remained stable with the cell sheet completely covering it, and no detachment or aggregation was observed. On the other hand, in Test Examples 1-3 and 1-4, the cell sheet did not detach from the culture dish, and the cells did not form aggregates to form a sheet. Furthermore, even on the 8th day of culture, areas with no cells or low cell density were observed on the culture dish in Test Examples 1-3 and 1-4, and cell sheet formation was not observed. Some areas also showed detachment or aggregation, as shown in the photograph.

As described above, while it is generally believed that seeding more cells is advantageous for shortening the culture period, the unexpected result was obtained that the conditions in Experimental Examples 1-2 were optimal seeding conditions for forming self-multilayered tissue.

In the above experiment, Cellmatrix I (manufactured by Nitta Gelatin Co., Ltd.) was used as the type I collagen applied to the culture dish, but it was confirmed that similar results could be obtained with the following type I collagens and recombinant peptides.
• Corning BioCoat (manufactured by Corning)
• Collagen I-coated dish (manufactured by IWAKI Corporation)
• cellnest Human Type I Collagen-like Recombinant Peptide (manufactured by Fujifilm Corporation)

(Test example 2)
<Test Example 2-1>
The growth curve was evaluated in the same manner as in Test Example 1-2. The results are shown in Figure 3.

<Test Example 2-2>
Except for replacing dental pulp stem cells with bone marrow stem cells, one of the representative MSCs used in medical science, the growth curve evaluation was performed in the same manner as in Experiment 2-1. The results are shown in Figure 3.

<Test Example 2-3>
Except for replacing dental pulp stem cells with umbilical cord stem cells, one of the representative MSCs used in medical science, the growth curve evaluation was performed in the same manner as in Experiment 2-1. The results are shown in Figure 3.

As shown in Figure 3, both Test Example 2-2 (marked with "●" in Figure 3) and Test Example 2-3 (marked with "▲" in Figure 3) showed lower cell proliferation and no self-multilayering compared to Test Example 2-1 (marked with "□" in Figure 3).

Figure 4 shows phase-contrast microscope images of test examples 2-1 to 2-3 on day 10 of culture.
In Test Example 2-1, a stable self-multilayered cell sheet was formed. On the other hand, in Test Example 2-2, although confluence was reached, self-multilayering was not observed. In Test Example 2-3, the cells detached from the culture dish and formed aggregates.

[Cell Sheet]
Figure 5 shows stereomicroscope images of cell sheets on day 10 of culture for Test Examples 2-1 and 2-2, and Figure 6 shows a stereomicroscope image of cell sheets on day 10 of culture for Test Example 2-3.
The autologous multilayered cell sheet of dental pulp stem cells in Test Example 2-1 was a robust cell sheet that did not tear even when grasped with sharp, precision tweezers, exhibiting remarkable operability and moldability. On the other hand, in the case of bone marrow stem cells in Test Example 2-2, the cells had only reached confluence and had not formed a sheet, making mechanical grasping impossible. In the case of umbilical cord stem cells in Test Example 2-3, aggregates (arrows in Figure 6) were observed after detachment from the culture dish.

Each cell sheet from Test Examples 2-1 to 2-3, cultured on day 10, was fixed with 4% paraformaldehyde (manufactured by Kanto Chemical Co., Ltd.) for 3 days and then cryopreserved in optimal cutting temperature compound (manufactured by Sakura Finetech Japan Co., Ltd.).
Frozen sections were prepared according to standard procedures and stained with hematoxylin and eosin (HE) and Masson's trichrome (MT). The results are shown in Figure 7 (upper panel: HE staining, lower panel: MT staining).
Test example 2-1 showed a multilayer cell structure due to the production of abundant collagen fibers, which was positive for aniline blue by MT staining. On the other hand, test example 2-2 was negative for aniline blue and showed a monolayer structure without collagen production, and test example 2-3 showed cell aggregation.

Each cell sheet from Test Examples 2-1 to 2-3, cultured on day 10, was fixed with 4% paraformaldehyde (manufactured by Kanto Chemical Co., Ltd.) and cryopreserved in optimal cutting temperature compound (manufactured by Sakura Finetech Japan Co., Ltd.).
Frozen sections were prepared according to standard procedures, and stained with TUNEL (Roche Diagnostics) and DAPI (Nacalai Tesque) according to the manufacturer's recommended methods. The results are shown in Figure 8 (top panel: TUNEL staining, middle panel: DAPI staining, bottom panel: merged sections).
Both Test Examples 2-1 and 2-2 were TUNEL-negative. On the other hand, many TUNEL-positive cells were observed in the aggregates of Test Example 2-3, confirming cell death.

Furthermore, to analyze the percentage of dead cells in the cell sheets, each cell sheet from Test Example 2-1 and Test Example 2-2 was stained using the Live or Dead Cell Viability Assay Kit (AAT Bioquest), and the number of dead cells was quantitatively evaluated. Each cell type was seeded in a 12-well plate under the conditions of Test Example 2-1 or 2-2, and on day 10 of culture, 10 fields of view (400x magnification) were randomly selected from each well to evaluate for dead cells. As a result, the percentage of dead cells in Test Example 2-1 was 1.24 ± 0.006%, and the percentage of dead cells in Test Example 2-2 was 1.16 ± 0.007%, indicating that only a very small number of dead cells were observed.

Figure 9 shows the results of immunofluorescence staining of various collagens in the autologous multilayer cell sheets of dental pulp stem cells from Test Example 2-1 on day 10 of culture. Note that bone marrow stem cells from Test Example 2-2 did not form sheets and did not produce collagen, and umbilical cord stem cells from Test Example 2-3 formed aggregates and did not form sheets, so these were excluded from this analysis.
In Figure 9, the left column shows the results of staining various types of collagen (top row: type I collagen, middle row: type IV collagen, bottom row: type VII collagen), the center column shows the results of DAPI staining, and the right column shows the results of merging. The primary antibodies used are as follows.
• Anti-type I collagen antibody: collagen type I alpha 1 (manufactured by Cell Signaling Technology)
• Anti-type IV collagen antibody: collagen type IV (manufactured by Abcam)
• Anti-Type VII collagen antibody: collagen type VII (manufactured by Abcam)

As shown in Figure 9, in Test Example 2-1, type I, type IV, and type VII collagen were positive throughout all layers of the cell sheet. These collagens are major constituent proteins of the extracellular matrix (ECM) in vivo and are thought to confer strength and flexibility (malleability) to the self-multilayered cell sheet.

The mechanical strength of the autologous multilayered cell sheet (hereinafter referred to as "cell sheet") of dental pulp stem cells cultured for 10 days in Test Example 2-1 was measured as follows.
- Compressive Strength -
A cell sheet was placed in a 5 mm diameter hole on the stage of a load-displacement measurement unit (FSA-0.5KE-5N, manufactured by IMADA Corporation). A load of 1 mm/min was applied using a 3 mm diameter measuring rod, and the load change until the cell sheet fractured was monitored and quantitatively evaluated.
As a result, the compressive strength of the cell sheet was 0.028 ± 0.008 N.

(Test example 3)
[Cell tubes]
The autologous multilayer cell sheet of dental pulp stem cells cultured for 10 days in Test Example 2-1 was rolled up (see Figure 10). The rolled structure was cultured at 37°C in a 4.7% CO2 environment using cGMP xeno-/serum- _free culture medium (XFM) (PRIME-XV® MSC Expansion XSFM; manufactured by Irvine Scientific Corporation/Fujifilm Wako Pure Chemical Corporation). Figure 11 shows the state after 4 days of culture following roll formation.

Figure 12 shows the results of hematoxylin-eosin (HE) staining of a cross-section of a cell tube four days after culture following roll formation.
As shown in Figure 12, solid cell tubes were obtained, rather than hollow ones.

Figure 13 shows the results of immunofluorescence staining of various collagens on a cross-section of a cell tube cultured for 4 days after rolling.
In Figure 13, the left column shows the results of staining various types of collagen (top row: type I collagen, middle row: type IV collagen, bottom row: type VII collagen), the center column shows the results of DAPI staining, and the right column shows the results of merging. The primary antibodies used are as follows.
• Anti-type I collagen antibody: collagen type I alpha 1 (manufactured by Cell Signaling Technology)
• Anti-type IV collagen antibody: collagen type IV (manufactured by Abcam)
• Anti-Type VII collagen antibody: collagen type VII (manufactured by Abcam)

As shown in Figure 13, type I, type IV, and type VII collagen were positive throughout the entire cell tube on day 4 of culture after roll-up.

The mechanical strength of the cell tubes on day 4 of culture after being rolled was measured as follows.
-Tensile Strength-
The cell tube was grasped using the force gauge (measuring device) and dedicated jig (clip) of a load-displacement measuring unit (FSA-0.5KE-5N, manufactured by IMADA Corporation), and the load change until the cell tube ruptured was monitored and quantitatively evaluated by pulling it upward at a speed of 1 mm/min.
As a result, the tensile strength of the cell tube was 0.38 ± 0.130 N.

(Test example 4)
[Cell disk]
<Test Example 4-1>
Dental pulp stem cells were cultured in the same manner as in Test Example 2-1, except that the culture period was set to one month.

<Test Example 4-2>
Bone marrow stem cells were cultured in the same manner as in Test Example 2-2, except that the culture period was set to one month.

Figure 14 shows phase-contrast microscope images of cells after one month of culture for test examples 4-1 and 4-2.
Test example 4-1 formed a stable, self-multilayered cell sheet. On the other hand, test example 4-2 showed increased thickness and a multilayered appearance compared to the confluent state of test example 2-2.

Stereomicroscope images of cells from test examples 4-1 and 4-2 after one month of culture are shown in Figure 15 (before loading with tweezers) and Figure 16 (after loading with tweezers).
As shown in Figures 15 and 16, the autologous multilayered cell sheet of dental pulp stem cells in Test Example 4-1 was a robust cell sheet that did not tear even when grasped with sharp, precision tweezers, and was disc-shaped with increased thickness and strength compared to the cell sheet in Test Example 2-1. On the other hand, the bone marrow stem cell sheet in Test Example 4-2 tore when touched with tweezers, making it impossible to grasp mechanically.

Cells from Test Examples 4-1 and 4-2, cultured for one month, were fixed with 4% paraformaldehyde (manufactured by Kanto Chemical Co., Ltd.) for three days and then cryopreserved in optimal cutting temperature compound (manufactured by Sakura Finetech Japan Co., Ltd.).
Frozen sections were prepared according to standard procedures and stained with hematoxylin and eosin (HE) and Masson's trichrome (MT). The results are shown in Figure 17 (HE staining) and Figure 18 (MT staining).
Test example 4-1 showed no dead cells and the cells were in good condition, exhibiting a multilayer cell structure due to the production of abundant collagen fibers that were positive for aniline blue. On the other hand, in test example 4-2, although multilayering due to the production of collagen fibers that were positive for aniline blue was observed, many cells had died, and the presence of cells was extremely sparse.

Cells from Test Examples 4-1 and 4-2, cultured for one month, were stained using TUNEL (Roche Diagnostics) and DAPI (Nacalai Tesque) according to the manufacturer's recommended method. The results are shown in Figure 19 (top panel: TUNEL staining, middle panel: DAPI staining, bottom panel: merged).
Test example 4-1 was TUNEL-negative and showed no cell death. On the other hand, in test example 4-2, many cells had already died, and TUNEL-positive cell death was confirmed in the observed cells.

Figure 20 shows the results of immunofluorescence staining of various collagens in dental pulp stem cells after one month of culture in Test Example 4-1.
In Figure 20, the left column shows the results of staining various types of collagen (top row: type I collagen, middle row: type IV collagen, bottom row: type VII collagen), the center column shows the results of DAPI staining, and the right column shows the results of merging. The primary antibodies used are as follows.
• Anti-type I collagen antibody: collagen type I alpha 1 (manufactured by Cell Signaling Technology)
• Anti-type IV collagen antibody: collagen type IV (manufactured by Abcam)
• Anti-Type VII collagen antibody: collagen type VII (manufactured by Abcam)

As shown in Figure 20, in Test Example 4-1, type I, type IV, and type VII collagen were positive throughout all layers of the cell sheet.
On the other hand, in Test Example 4-2, many cells had died and the ECM itself had undergone degenerative changes, so the staining of various collagens was negative or showed only weakly positive results, much weaker than in Test Example 4-1.

The mechanical strength of the autologous multilayered cell sheet (hereinafter referred to as "cell disc") of dental pulp stem cells in a disc-like state after one month of culture in Test Example 4-1 was measured as follows.
- Compressive Strength -
A cell disc was placed in a 5 mm diameter hole on the stage of a load-displacement measurement unit (FSA-0.5KE-5N, manufactured by IMADA Corporation). A load of 1 mm/min was applied using a 3 mm diameter measuring rod, and the load change until the cell disc fractured was monitored and quantitatively evaluated.
As a result, the compressive strength of the cell disk was 0.75 ± 0.175 N.

-Tensile Strength-
The cell disc was grasped using the force gauge (measuring device) and dedicated jig (clip) of a load-displacement measuring unit (FSA-0.5KE-5N, manufactured by IMADA Corporation), and the load change until the cell disc fractured was monitored and quantitatively evaluated while being pulled upward at a speed of 1 mm/min.
As a result, the tensile strength of the cell disk was 0.32 ± 0.155 N.

(Test example 5)
<Test Example 5-1>
The proliferation curve was evaluated in the same manner as in Experimental Example 1, except that the seeding rate of dental pulp stem cells was set to 0.75 × ^10⁴ cells/ ^cm² . The results are shown in Figure 21.

<Test Example 5-2>
Except for setting the seeding rate of dental pulp stem cells to 1.5 × ^10⁴ cells/ ^cm² , the proliferation curve was evaluated in the same manner as in Experimental Example 1. The results are shown in Figure 21.

As shown in Figure 21, Test Example 5-2 (marked with "■" in Figure 21) showed proliferation similar to that of Test Example 1-2, and Test Example 5-1 (marked with "●" in Figure 21) showed cell proliferation equivalent to that of Test Example 1-1.

[Cell Sheet]
For test examples 5-1 to 5-2, phase-contrast microscope images of the cell sheets on day 10 of culture are shown in Figure 22, and stereomicroscope images are shown in Figure 23.
Test examples 5-1 to 5-2 showed a dense, multi-layered arrangement of cells, and all could be picked up with tweezers.

Cells from Test Examples 5-1 and 5-2, cultured to day 10, were fixed with 4% paraformaldehyde (manufactured by Kanto Chemical Co., Ltd.) and cryopreserved in optimal cutting temperature compound (manufactured by Sakura Finetech Japan Co., Ltd.).
Frozen sections were prepared according to standard procedures and stained with hematoxylin-eosin (HE) and Masson's trichrome (MT). The results are shown in Figure 24 (upper panel: HE staining, lower panel: MT staining).
Test example 5-1 formed a densely structured material composed of cells and collagen histologically, but did not show significant multilayering. On the other hand, test example 5-2 formed a structure in which cells and collagen showed significant multilayering.

Furthermore, to analyze the percentage of dead cells in the cell sheets, each cell sheet from Test Example 5-1 and Test Example 5-2 was stained using the Live or Dead Cell Viability Assay Kit (AAT Bioquest), and the number of dead cells was quantitatively evaluated. Each cell type was seeded in a 12-well plate under the conditions of Test Example 5-1 or 5-2, and on day 10 of culture, 10 fields of view (400x magnification) per well were randomly selected to evaluate dead cells. As a result, the percentage of dead cells in Test Example 5-1 was 0.51 ± 0.003%, and the percentage of dead cells in Test Example 5-2 was 1.33 ± 0.010%, indicating that only a very small number of dead cells were observed.

Figure 25 shows the results of immunofluorescence staining of various collagens in the cell sheet of dental pulp stem cells cultured on day 10 in Test Example 5-1, and Figure 26 shows the results of immunofluorescence staining of various collagens in the cell sheet of dental pulp stem cells cultured on day 10 in Test Example 5-2.
In Figures 25-26, the left column shows the results of staining various types of collagen (top row: type I collagen, middle row: type IV collagen, bottom row: type VII collagen), the center column shows the results of DAPI staining, and the right column shows the results of merging. The primary antibodies used are as follows.
• Anti-type I collagen antibody: collagen type I alpha 1 (manufactured by Cell Signaling Technology)
• Anti-type IV collagen antibody: collagen type IV (manufactured by Abcam)
• Anti-Type VII collagen antibody: collagen type VII (manufactured by Abcam)

As shown in Figures 25-26, in Test Examples 5-1 and 5-2, type I, type IV, and type VII collagen were positive throughout all layers of the cell sheet. These collagens are major constituent proteins of the extracellular matrix (ECM) in vivo and are thought to confer strength and flexibility (malleability) to the self-multilayered cell sheet.

(Test example 6)
Under seeding conditions that resulted in the formation of self-multilayered cell sheets that could be grasped with tweezers on day 10 of culture, the cell sheet thickness was measured and quantitatively evaluated for Test Example 2-1, which had the maximum cell sheet thickness, and Test Example 5-1, which had the minimum cell sheet thickness.
Thickness was measured using HE-stained tissue photographs. Ten locations on the cell sheet were randomly selected, and their thickness was measured.
As a result, the cell sheet thickness of Test Example 2-1 was 33.0 ± 6.16 μm, and the cell sheet thickness of Test Example 5-1 was 8.0 ± 2.20 μm. The cell sheet of Test Example 2-1 was more than three times thicker than the cell sheet of Test Example 5-1.

As described above, according to the present invention, the culture period required for the production of cell sheets, which are multilayer cell structures, can be shortened, cell death in the multilayer cell structures can be suppressed, and furthermore, multilayer cell structures of various shapes can be produced.

This application claims priority based on Japanese Patent Application No. 2022-064720, filed on 8 April 2022, and the entire contents of Japanese Patent Application No. 2022-064720 are incorporated herein by reference.

Claims (10)

This includes culturing mesenchymal stem cells derived from dental pulp in a serum-free medium using culture equipment coated with type I collagen or its fragments,
A method for producing a multilayer cell structure, characterized in that the seeding number of mesenchymal stem cells derived from dental pulp is 0.75 × ^10⁴ cells/ ^cm² or more and 1.5 × ^10⁴ cells/ ^cm² or less. The method for producing a multilayer cell structure according to claim 1, wherein the serum-free culture medium does not contain components from a different animal species. A method for producing a multilayer cell structure according to any one of claims 1 to 2, wherein the shape of the multilayer cell structure is one of a sheet, a rod, or a disc. The method for producing a multilayer cell structure according to claim 3, wherein the shape of the multilayer cell structure is sheet-like and has a thickness of 5 μm to 40 μm. A method for producing a multilayer cell structure according to any one of claims 1 to 2, wherein the proportion of dead cells in the multilayer cell structure is 5% or less. A method for producing a multilayer cell structure according to any one of claims 1 to 2, wherein the multilayer cell structure expresses type I collagen, type IV collagen, and type VII collagen. A multilayer cell structure of dental pulp-derived mesenchymal stem cells produced by the method for producing a multilayer cell structure described in claim 1 ,
A multilayer cell structure characterized in that the proportion of dead cells in the multilayer cell structure is 5% or less. The multilayer cell structure according to claim 7, wherein the multilayer cell structure expresses type I collagen, type IV collagen, and type VII collagen. The multilayer cell structure according to any one of claims 7 to 8, wherein the shape of the multilayer cell structure is one of a sheet, a rod, or a disc. The multilayer cell structure according to claim 9, wherein the shape of the multilayer cell structure is sheet-like and has a thickness of 5 μm to 40 μm.