WO2024024561A1 - Method for producing keratinocytes, medium kit for continuously culturing keratinocytes, and keratinocytes - Google Patents

Abstract

This method for producing keratinocytes includes a separation step that is repeated multiple times, in which step trypsinization is performed on a cell population including keratinocytes induced from pluripotent stem cells, to separate keratinocytes. The present invention realizes a technique of producing a cell population of keratinocytes induced from pluripotent stem cells, the cell population including keratinocytes having high proliferation capacity.

Description

Method for producing keratinocytes, medium kit for continuous culture of keratinocytes, and keratinocytes

The present invention relates to a method for producing keratinocytes, a medium kit for continuously culturing keratinocytes, and keratinocytes.

In recent years, attempts have been made to induce differentiation of pluripotent stem cells such as ES cells and iPS cells to produce various types of tissues and cells. As an example, Non-Patent Documents 1 and 2 describe techniques for inducing keratinocytes from pluripotent stem cells.

Res Ther 2014;5:60-70. Proc Natl Acad SciUSA 2006;103:1792-7.

However, keratinocytes produced by the techniques described in Non-Patent Documents 1 and 2 have a lower proliferation ability and a shorter lifespan than those produced by culturing human primary cells. Furthermore, in the techniques described in Non-Patent Documents 1 and 2, a method of introducing an immortalizing gene is used, and the obtained keratinocytes are not suitable for transplantation. As described above, a technique for producing keratinocytes suitable for transplantation from pluripotent stem cells has not been established.

One aspect of the present invention has been made in view of the above-mentioned problems, and its purpose is to realize a technology for producing a cell population of keratinocytes derived from pluripotent stem cells, including keratinocytes with high proliferative ability. It is in.

In order to solve the above problems, a method for producing keratinocytes according to one aspect of the present invention includes separation in which keratinocytes are separated by treating a cell population containing keratinocytes derived from pluripotent stem cells with trypsin. Perform the process multiple times.

A method for producing keratinocytes according to another aspect of the present invention includes a culturing step of continuously culturing keratinocytes induced from pluripotent stem cells in the presence of feeder cells, a Rho kinase inhibitor, and an extracellular matrix.

In a method for producing keratinocytes according to another aspect of the present invention, a cell population containing keratinocytes induced from pluripotent stem cells is subjected to a separation step in which keratinocytes are separated by treatment with trypsin multiple times. Keratinocytes are continuously cultured in the presence of feeder cells, Rho kinase inhibitor and extracellular matrix.

A medium kit for continuous culture of keratinocytes derived from pluripotent stem cells according to one aspect of the present invention includes feeder cells, a Rho kinase inhibitor, and an extracellular matrix.

Keratinocytes according to one aspect of the present invention are derived from pluripotent stem cells and have a cell population doubling level (PDL) of more than 15.

According to one aspect of the present invention, it is possible to provide a technique for producing a cell population of keratinocytes derived from pluripotent stem cells, including keratinocytes with high proliferative ability.

FIG. 3 is a diagram showing a trypsin treatment step in a method for producing keratinocytes according to one embodiment of the present invention. It is a figure showing the cell morphology of a control group and a trypsin-treated group of hESC-derived keratinocytes in Examples. FIG. 3 is a diagram showing the morphology of cells recovered by the first trypsin treatment in Examples. FIG. 3 is a diagram showing the colony forming effect of hESC-derived keratinocytes in Examples. FIG. 2 is a diagram showing the morphology of hESC-derived keratinocytes cultured under continuous culture conditions and fibroblast culture conditions in Examples. FIG. 2 is a diagram showing the results of gene expression analysis of hESC-derived keratinocytes in Examples. FIG. 3 is a diagram showing the results of airlift culture of hESC-derived keratinocytes in Examples. FIG. 3 shows marker expression in epithelial equivalents derived from hESC-derived keratinocytes in Examples. FIG. 2 is a diagram showing the results of continuous culture of hESC-derived keratinocytes in Examples. FIG. 2 is a diagram showing a growth curve of hESC-derived keratinocytes during continuous culture in Examples. FIG. 2 is a diagram showing the doubling time of hESC-derived keratinocytes during continuous culture in Examples. FIG. 3 is a diagram showing the cell morphology of hESC-derived keratinocytes cultured under five culture conditions in Examples. It is a figure showing the cell density of hESC-derived keratinocytes cultured under five culture conditions in Examples. FIG. 2 is a diagram showing the doubling time of hESC-derived keratinocytes cultured under culture conditions 1 and 2 in Examples.

[Method for producing keratinocytes]
In a method for producing keratinocytes according to one embodiment of the present invention, a cell population containing keratinocytes induced from pluripotent stem cells is subjected to a separation step of separating keratinocytes by treating with trypsin multiple times.

As used herein, "pluripotent stem cells" may be any undifferentiated cells that have the ability to differentiate into at least keratinocytes. Keratinocytes are the main cells constituting the epidermis, are also called epidermal keratinocytes, and have the ability to proliferate and differentiate. The pluripotent stem cells may be human pluripotent stem cells. Examples of pluripotent stem cells include embryonic stem cells (ES cells) isolated from early embryos, embryonic germ cells (EG cells) isolated from primordial germ cells during the fetal period, Germline stem cells (GS cells) isolated from testis immediately after birth, stem cells derived from bone marrow such as iliac bone marrow and jawbone bone marrow, mesenchymal stem cells such as stem cells derived from adipose tissue, and skin cells. By introducing multiple genes into the somatic cells of the subject, the dedifferentiation of the subject's own somatic cells is induced, resulting in somatic cell-derived induced pluripotent stem cells (or induced pluripotent stem cells) that have pluripotency similar to ES cells. Examples include induced pluripotent stem cells (iPS cells).

(separation process)
In the separation step, keratinocytes are separated from the cell population obtained in the process of inducing differentiation of pluripotent stem cells. The cell population obtained in the process of inducing differentiation of pluripotent stem cells may contain many other cell types in addition to keratinocytes, and may be a miscellaneous cell population. Such a cell population may be obtained during the process of inducing differentiation of pluripotent stem cells into keratinocytes, or may be obtained during the process of inducing differentiation of pluripotent stem cells into somatic cells such as hepatocytes and muscle cells. It may be obtained from

A cell population containing keratinocytes derived from pluripotent stem cells may be obtained by a conventionally known differentiation induction method. The method for producing keratinocytes according to one embodiment of the present invention may include a differentiation step of inducing differentiation of pluripotent stem cells to obtain a cell population containing keratinocytes.

In one example, the cell population containing keratinocytes derived from pluripotent stem cells may be a cell population obtained when inducing differentiation of hepatocytes or hepatic progenitor cells from pluripotent stem cells. Examples of methods for obtaining such cell populations include methods of inducing differentiation of hepatocytes and hepatic progenitor cells through forming embryoid bodies from pluripotent stem cells (see also International Publication No. WO2019/131938, etc.). (see ). When inducing differentiation of hepatocytes and hepatic progenitor cells, it is preferable to add a hepatic differentiation-inducing factor to the culture medium and perform the culture. Examples of hepatic differentiation-inducing factors include cytokines and cell growth factors such as hepatocyte growth factor (HGF), Wnt, and R-spondin 1; Rho kinase inhibitors such as Y27632; laminin (or its fragment), collagen, fibronectin, Examples include extracellular matrices such as Matrigel; MAPK inhibitors; and the like, and these may be used alone or in combination.

It is preferable to induce a cell population containing keratinocytes by adhesion culture. Adhesive culture is performed, for example, in a container coated with an extracellular matrix as described above, or by co-cultivation with feeder cells. In addition, selective pressure may be applied by adding an antibiotic to the medium and culturing. Examples of antibiotics include penicillin, streptomycin, amphotericin B, etc., and these may be used alone or in combination.

In the separation step, a cell population containing keratinocytes derived from pluripotent stem cells is treated with trypsin. Keratinocytes are separated from the cell population by performing such a separation step including trypsin treatment multiple times. Trypsin treatment is performed under conditions that allow separation of keratinocytes from the cell population. In trypsin treatment, for example, keratinocytes are separated from a cell population by peeling the cell population adhered to a container such as a culture container for culturing cells from the container. In the trypsin treatment in the separation step, for example, a trypsin-EDTA solution is used.

In the separation step, keratinocytes are separated based on differences in cell adhesiveness after trypsin treatment. Keratinocytes have strong cell-to-cell adhesion and ability to adhere to extracellular matrix. Therefore, it is assumed that keratinocytes derived from pluripotent stem cells also have higher adhesive properties than other cells. In the separation step, for example, out of the cell population adhered to the container, cells with lower adhesion than keratinocytes are detached from the container by trypsin treatment and removed, thereby separating keratinocytes from the cell population. Keratinocytes separated from the cell population may be detached from the container by further treatment with trypsin.

In the separation step, the cell population including keratinocytes that have adhered to the container is subjected to a first trypsin treatment to detach cells other than keratinocytes from the container, and after the first trypsin treatment, the cell population that has adhered to the container is treated with keratinocytes. The keratinocytes are separated by performing a second trypsin treatment to detach them from the container. In the separation step, as shown in FIG. 1, keratinocytes are separated from the cell population by multiple trypsin treatments. FIG. 1 is a diagram showing a trypsin treatment step in a method for producing keratinocytes according to one embodiment of the present invention. Note that some keratinocytes may be peeled off during the separation step. Furthermore, if all cells other than keratinocytes are not removed in one separation step, the purity of the keratinocytes can be increased by performing the separation step multiple times using cells passaged after the separation step.

As shown in FIG. 1, in the separation step, trypsin is first added to a container to which a cell population containing keratinocytes derived from pluripotent stem cells has adhered, and a first trypsin treatment is performed. In the first trypsinization, the cell population adhered to the container is incubated in trypsin.

The first trypsin treatment is trypsin treatment of a cell population including keratinocytes adhered to the container under treatment conditions that peel cells other than keratinocytes from the container. The first trypsin treatment causes cells other than keratinocytes to detach from the container and float in the supernatant, while the keratinocytes remain attached to the container. The cells that detach from the container upon the first trypsin treatment may be less adherent cells than keratinocytes.

The trypsin concentration in the first trypsin treatment may be within a concentration range in which cells other than keratinocytes are detached and keratinocytes are not detached in the first treatment step, and the commonly used 0.25 w/v% trypsin 1 mmol/L EDTA solution That's fine. The temperature of trypsin in the first trypsin treatment may be within a temperature range in which cells other than keratinocytes are detached but keratinocytes are not detached, and is preferably room temperature or higher, and more preferably 37°C. However, it is desirable to keep in mind that changing the concentration and temperature of the trypsin-EDTA solution will change the enzyme activity and will change the appropriate treatment time.

In the first trypsin treatment, a cell population containing keratinocytes is exposed to trypsin for at least 1 minute and at most 3 minutes. In the first trypsin treatment, the time for exposing the cell population to trypsin may be within a range in which cells other than keratinocytes are detached but keratinocytes are not detached, and is preferably 3 minutes. As an example, when the temperature is 37°C, by exposure to trypsin 0.25 w/v% 1 mmol/L EDTA solution for 3 minutes, cells other than keratinocytes detach from the container, but most keratinocytes adhere to the container. It remains as it is.

Note that by the first trypsin treatment, some of the keratinocytes adhered to the inside of the container may be peeled off. In this case, it is preferable that more keratinocytes remain adhered to the container than keratinocytes floating in the supernatant in the container after the first trypsin treatment. Additionally, the first trypsin treatment and subsequent removal of the supernatant may be repeated multiple times before the second trypsin treatment. This further reduces the amount of cells other than keratinocytes in the container and increases the purity of the keratinocytes.

Next, remove the supernatant in the container after the first trypsin treatment. The cells detached from the container by the first trypsin treatment are suspended in the supernatant after the first trypsin treatment, and by removing the supernatant, the cells detached by the first trypsin treatment are removed from the container. Cells including keratinocytes that were not detached in the first trypsin treatment adhered to the container from which the supernatant was removed.

The second trypsin treatment is a trypsin treatment of the cell population adhered to the container after the first trypsin treatment under treatment conditions to peel the keratinocytes from the container. The keratinocytes can be separated by collecting the keratinocytes exfoliated from the container by the second trypsin treatment. In the second trypsinization, the cell population that adhered to the container after the first trypsinization is incubated in trypsin.

Due to the second trypsin treatment, keratinocytes are detached from the container and suspended in the supernatant. The second trypsin treatment may cause cells that are more adhesive than keratinocytes to remain attached to the container. Further, the supernatant in the container after the second trypsin treatment may contain other cells together with keratinocytes. In this case, it is preferable that the amount of keratinocytes contained in the supernatant is larger than that of other cells.

The trypsin concentration in the second trypsin treatment may be within a concentration range that allows the keratinocytes to detach, and may be a commonly used 0.25 w/v% trypsin 1 mmol/L EDTA solution. The temperature of trypsin in the second trypsin treatment may be within a temperature range at which keratinocytes are exfoliated, preferably room temperature or higher, and more preferably 37° C. or higher.

In the second trypsin treatment, the cell population from which detached cells have been removed in the first trypsin treatment is exposed to trypsin for 3 minutes or more. In the second trypsin treatment, the time for exposing the keratinocytes to trypsin may be within a range that allows the keratinocytes to be exfoliated, and is more preferably 3 minutes or more, 6 minutes or more, or 9 minutes or more. As an example, when the temperature is 37° C., keratinocytes are detached from the container by exposing the cell population to a 0.25 w/v% trypsin, 1 mmol/L EDTA solution for at least 3 minutes and no more than 7 minutes.

Note that in the first trypsin treatment and the second trypsin treatment, the concentration and temperature of trypsin may be the same, and only the trypsin treatment time for exposure to trypsin may be changed. In this case, the processing time of the first trypsin treatment may be made shorter than the processing time of the second trypsin treatment.

By separating keratinocytes from a cell population containing keratinocytes derived from pluripotent stem cells through the separation step, the resulting keratinocytes are of high purity and a variety of keratinocytes can be obtained. Therefore, the obtained keratinocytes may include keratinocytes with low proliferative ability, but are also likely to include keratinocytes with high proliferative ability. By obtaining such a variety of keratinocytes, it is possible to culture them and select keratinocytes with high proliferation ability.

With conventional purification methods using cloning rings, the number of cells that can be recovered is extremely limited, so there is a possibility that keratinocytes with high proliferative ability cannot be recovered. Furthermore, in order to separate multiple colonies using cloning rings, as many cloning rings as the number of colonies to be separated are required. According to the method for producing keratinocytes according to one embodiment of the present invention, cells that have been induced to differentiate as keratinocytes can be collected from the entire culture dish, and keratinocytes with high proliferative ability can be easily and stably produced. Can be done. With conventional techniques, keratinocytes induced from pluripotent stem cells had low proliferation ability, but according to the method for producing keratinocytes according to one embodiment of the present invention, keratinocytes with high proliferation ability can be stably produced. Even keratinocytes derived from pluripotent stem cells can be continuously cultured for long periods of time.

(Culture process)
A method for producing keratinocytes according to another aspect of the present invention includes a culturing step of continuously culturing keratinocytes induced from pluripotent stem cells in the presence of feeder cells, a Rho kinase inhibitor, and an extracellular matrix. The keratinocytes in the culture step may be keratinocytes separated from the cell population derived from pluripotent stem cells by the above-described separation step. That is, a cell population containing keratinocytes derived from pluripotent stem cells is subjected to a separation step in which keratinocytes are separated by trypsin treatment multiple times, and the separated keratinocytes are treated with feeder cells, a Rho kinase inhibitor, and extracellular Also included in the scope of the present invention is a method for producing keratinocytes that is continuously cultured in the presence of a substrate. Note that the above-described separation step may be performed during continuous culture to purify keratinocytes.

As used herein, "culture" refers to growing cells in vitro. Cultivating cells using a medium refers to culturing cells in a state where the medium and cells are in contact, such as culturing cells in a medium or culturing cells on a medium. The medium may be in gel form or liquid form.

The medium used in the culture step may be any medium that can culture keratinocytes, and conventionally known keratinocyte culture media can be used. As such a culture medium, a commercially available medium can be used, and one example is keratinocyte serum-free medium (DK-SFM). The keratinocyte culture medium may contain other known components for culturing keratinocytes.

In the culturing step, keratinocytes in a keratinocyte culture medium are cultured in the presence of feeder cells, a Rho kinase inhibitor, and an extracellular matrix. As an example, keratinocytes are seeded on a feeder layer containing feeder cells and co-cultured in a keratinocyte culture medium containing a Rho kinase inhibitor and extracellular matrix. In the culturing process, the extracellular matrix may be coated onto the container in which the keratinocytes are cultured.

In the culture step, by continuously culturing keratinocytes in the presence of feeder cells, a Rho kinase inhibitor, and an extracellular matrix, it is possible to culture keratinocytes that maintain their proliferation ability, and continuous culture for a long period of time can be achieved.

Feeder cells are cells that act as a basal layer for keratinocytes and provide secreted factors, extracellular matrix, and cell contacts to maintain keratinocytes. The feeder cells may be prepared by being inactivated by gamma irradiation or the like. As the feeder cells, conventionally known feeder cells for culturing keratinocytes can be used, and one example is mouse embryonic fibroblasts. The feeder cells can be used as feeder layers prepared in layers. The amount of feeder cells used in the feeder layer may be any amount suitable for continuous culture of keratinocytes, and as an example, it is 2.0 × 10 ⁴ or more and 2.5 × 10 ⁴ or less per 1 cm ² . The number is preferably 2.5×10 ^{4 , and more preferably 2.5×10 4} .

Rho kinase inhibitors inhibit the function of Rho kinase, an intracellular phosphorylating enzyme, and suppress cell death. As the Rho kinase inhibitor, conventionally known Rho kinase inhibitors can be used, and one example is Y27632.

The Rho kinase inhibitor can be used by being added to a medium for culturing keratinocytes, and may be added in an amount suitable for continuous culturing of keratinocytes. As an example, the amount of Rho kinase inhibitor added is 10 μmol/L.

The extracellular matrix serves as a scaffold for cell culture and is a protein involved in cell adhesion, migration, proliferation, etc. As the extracellular matrix, conventionally known extracellular matrices can be used, and one example is laminin or a fragment thereof. Laminin may be produced from laminin-expressing cells by a known method, or a commercially available product may be purchased.

The extracellular matrix can be used by coating a container for culturing keratinocytes, as long as it is coated in an amount suitable for continuous culturing of keratinocytes. As an example, the amount of extracellular matrix to be coated is preferably 0.1 mL or more of a 2.5 μg/mL laminin-511E8 fragment solution per 1 cm ² of area to be coated on the container.

The culture conditions in the culture step may be any conventionally known culture conditions for keratinocytes. In the culturing step, for example, keratinocytes are cultured at a temperature of 30°C or higher and 40°C or lower, preferably 37°C. Further, in the culturing step, keratinocytes are cultured, for example, in the presence of 3% or more and 15% or less carbon dioxide, preferably in the presence of 5% carbon dioxide. Furthermore, in the culturing step, it is preferable to culture keratinocytes in a humidified environment; for example, keratinocytes are cultured in an environment with a humidity of 85% or more, preferably in an environment with a humidity of 95% or more.

By culturing keratinocytes derived from pluripotent stem cells in the presence of feeder cells, a Rho kinase inhibitor, and an extracellular matrix, it is possible to maintain proliferation ability and culture keratinocytes for a long period of time. With conventional technology, keratinocytes derived from pluripotent stem cells had low proliferative ability, but according to the method for producing keratinocytes according to one embodiment of the present invention, even keratinocytes derived from pluripotent stem cells can continue for a long period of time. Can be cultured.

Furthermore, keratinocytes produced by the method for producing keratinocytes according to one embodiment of the present invention can be distinguished from keratinocytes produced by other methods in that they have a high proliferation ability. Keratinocytes produced by the method for producing keratinocytes according to one embodiment of the present invention can be distinguished from other keratinocytes by differences in molecular markers or gene expression. A molecular marker for distinguishing keratinocytes produced by the keratinocyte production method according to one embodiment of the present invention from other keratinocytes is also included in the scope of the present invention.

[Medium kit for continuous culture of keratinocytes derived from pluripotent stem cells]
A medium kit for continuous culture of keratinocytes derived from pluripotent stem cells according to one aspect of the present invention includes feeder cells, a Rho kinase inhibitor, and an extracellular matrix. The media kit can be used with media for culturing keratinocytes derived from pluripotent stem cells. This allows keratinocytes to be cultured for a long period of time while maintaining their proliferation ability. The medium kit may further include a keratinocyte culture medium.

The feeder cells, Rho kinase inhibitor, and extracellular matrix contained in the culture medium kit are the same as those used in the culture step of the method for producing keratinocytes according to one embodiment of the present invention. Therefore, for details such as the types and contents of the feeder cells, Rho kinase inhibitor, and extracellular matrix included in the culture medium kit, and the culture method using these, please refer to the method for producing keratinocytes according to one embodiment of the present invention described above. The description of the culture process is incorporated herein by reference.

[Keratinocytes]
Keratinocytes according to one aspect of the invention are derived from pluripotent stem cells and have a population doubling level (PDL) of greater than 15. Furthermore, the keratinocytes according to one embodiment of the present invention have a cell population doubling time of 60 hours or less. The keratinocytes according to one embodiment of the present invention have high proliferative ability and maintain their proliferative ability even when continuously cultured.

Here, PDL is determined by dividing the number of cells in the cell population at the end of culture by the number of cells in the cell population at the start of culture. The keratinocytes according to one aspect of the present invention preferably have a PDL of 16 or more, 20 or more, 30 or more, 40 or more, or 45 or more.

The cell doubling time is intended to be the time required for the number of cells included in a cell population to double. The keratinocytes according to one aspect of the present invention preferably have a cell population doubling time of 55 hours or less, 50 hours or less, 45 hours or less, 40 hours or less, or 30 hours or less.

Keratinocytes according to one embodiment of the present invention can be produced by a method for producing keratinocytes according to one embodiment of the present invention. Furthermore, keratinocytes according to one embodiment of the present invention can be produced using a medium kit for continuous culture of keratinocytes induced from pluripotent stem cells according to one embodiment of the present invention.

The present invention is not limited to the embodiments described above, and various modifications can be made within the scope of the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. are also included within the technical scope of the present invention.

[1. Ethics statement]
All experiments using human cells and tissues were approved by the Ethics Review Committee of the National Institute of Biomedical Innovation. Informed consent was obtained from the patients' parents. The induction and culture of ESC lines is described in “the Guidelines for Derivation and Distribution of Human Embryonic Stem Cells (Notification of the Ministry of Education, Culture, Sports, Science, and Technology in Japan (MEXT), No. 156 of August 21, 2009 ; Notification of MEXT, No. 86 of May 20, 2010)” and “the Guidelines for Utilization of Human Embryonic Stem Cells (Notification of MEXT, No. 157 of August 21, 2009; Notification of MEXT, No. 87 of May 20 , 2010)”.

[2. Culture and differentiation of hESC]
SEES-2, a human embryonic stem cell (hESC) line established by the present inventors in past research, was normally cultured in an ESC culture medium with a feeder layer derived from mouse embryonic fibroblasts (see reference 1 below). ). Mouse embryonic fibroblasts for the maintenance of SEES-2 were isolated from ICR mouse fetuses on day 12.5 of gestation, cultured twice consecutively, and gamma-incubated with 30 Gy (Hitachi, MBR-1520 R-3). A feeder layer was prepared by irradiation. ESC medium was KO-DMEM (Thermo Fisher Scientific) with 20% KO-TM serum (KO-SR; Thermo Fisher Scientific), 2 mmol/L Glutamax-I (Thermo Fisher Scientific), and 0.1 mmol/L. Non-essential amino acids (NEAA; Thermo Fisher Scientific), 1 mmol/L sodium pyruvate (Thermo Fisher Scientific), and 50 ng/mL recombinant human full-length bFGF (Thermo Fisher Scientific) were added and adjusted.

To generate embryoid bodies (EBs), SEES-2 was exposed to the Rho kinase inhibitor Y-27632 (Fujifilm), followed by isolation into single cells with Accutase (Thermo Fisher Scientific) and 5 × ¹⁰ Cells/well were plated in 96-well plates in EB medium. As an EB medium, KO-DMEM was mixed with 20% KO-SR, 2 mmol/L glutamax-I, 0.1 mmol/L NEAA, 1 mmol/L pyruvic acid, and 1 mmol/L sodium pyruvic acid. A medium prepared by the following method was used.

The embryoid bodies were transferred to a 24-well plate coated with collagen type I and cultured in XF32 medium for 35 days. As XF32 medium, 15% knockout serum replacement XF CTS (Thermo Fisher Scientific), 2 mmol/L glutamax-I, 0.1 mmol/L NEAA, 1 mmol/L sodium pyruvate, 50 mg/mL l-ascorbic acid. KO-DMEM medium supplemented with diphosphate, 10 ng/mL heregulin-1b (R&D Systems, MN, USA), 200 ng/mL recombinant human IGF-1 (Sigma-Aldrich), and 20 ng/mL human bFGF. was used.

The differentiated cells were further co-cultured with a feeder layer of mouse embryonic fibroblasts, and grown in ESTEM-HE medium containing Wnt3a and R-spondin 1 (GlycoTechnica _, Kanagawa, Japan) (Reference 2, described below). When the culture medium became subconfluent, cells were harvested using 0.25 w/v% trypsin and 1 mmol/L EDTA (Fujifilm Wako Pure Chemicals) and placed in a 100 mm culture dish at a density of 1/4 confluence. I re-plated it. Thereafter, the medium was replaced every 3 days. Differentiated hESCs were continuously cultured and cryopreserved at the fourth generation.

[3. Purification and continuous culture of hESC-derived keratinocytes]
To prepare the feeder layer, 10% FBS (Thermo Fisher Scientific), 100 units/mL penicillin, 100 mg/mL streptomycin (Thermo Fisher Scientific), and 0.25 mg/mL amphotericin B (Bristol-Myers Squibb, Mouse fibroblasts derived from calvarial bone marrow were continuously cultured in α-modified Eagle's minimum essential medium (α-MEM: Thermo Fisher Scientific) containing (NY, USA). The feeder layer was prepared according to the method shown in a previous study (Reference 3, described below). Briefly, mouse fibroblasts treated with 10 mg/mL mitomycin C (Nacalai Tesque, Kyoto, Japan) for 2 hours were coated with LN-511-E8 (Nippi, Tokyo, Japan) according to the manufacturer's instructions. The cells were seeded at 2.5×10 ⁴ cells/cm ² in a culture dish prepared to prepare a feeder layer.

Cryopreserved differentiated hESCs were seeded and cultured at a density of 2.9 x ¹⁰⁴ cells/ ^cm2 on culture dishes coated with extracellular matrix LN-511-E8 (laminin 511E8). . The medium was keratinocyte serum-free medium (DK-SFM; Thermo Fisher Scientific) supplemented with 10 mmol/mL Y-27632, 100 units/mL penicillin, 100 mg/mL streptomycin, and 0.25 mg/mL amphotericin B. was used. Cultivation was performed by co-cultivating with a feeder layer in a humidified environment of 5% CO ₂ and 37°C. To isolate hESC-derived keratinocytes and purify the cell population, first and second trypsin treatments were performed before continuous culture in the 5th, 6th, and 7th generations.

As the first trypsin treatment, differentiated hESCs were treated with trypsin-EDTA for 3-6 minutes in an environment of 5% CO ₂ and 37°C. By removing the supernatant of this first trypsin treatment after incubation, cells that did not exhibit keratinocyte-like cell morphology in the supernatant were removed. After the first trypsin treatment, cells adhering to the culture dish were treated with a second trypsin for 3 to 7 minutes using trypsin-EDTA to isolate cells exhibiting keratinocyte-like cell morphology.

In the 6th generation, the proliferation of non-keratinocyte cells in differentiated hESCs was confirmed. Conventional culture conditions for fibroblasts using α-MEM medium containing 10% FBS, penicillin, and streptomycin, and DK-SFM medium containing Y-27632 and LN-511-E8 and a feeder layer were used. The cell suspension, which is the supernatant of the first trypsin treatment, was cultured under each continuous culture condition.

After reaching the 8th generation, hESC-derived keratinocytes were continuously cultured without additional trypsin treatment until the proliferation rate decreased. At the 13th generation, hESC-derived keratinocytes were also cultured in α-MEM medium containing 10% FBS to identify non-keratinocyte cells. The population doubling level (PDL) and doubling time of hESC-derived keratinocytes were calculated during continuous culture. In order to confirm the effect of trypsin treatment on cell separation, cells differentiated from SEES-2 were continuously cultured without trypsin treatment before continuous culture and used as a cell control group.

[4. Colony forming ability analysis of hESC-derived keratinocytes]
Seventh generation (PDL3.7) hESC-derived keratinocytes were seeded on a 6-well plate at a density of 100 cells/well as a control group and a trypsin-treated group, and keratinocytes were supplemented with 10 mmol/L of Y-27632. Co-cultured with a mouse feeder layer in culture medium (KCM). The composition of KCM followed that used in past studies (Reference 4, described below). Briefly, KCM was prepared using Dulbecco's modified Eagle's medium (SigmaeAldrich, MO, USA) with 100 units/mL penicillin, 100 mg/mL streptomycin, 0.25 mg/mL amphotericin B, 5% FBS, and 5 mg/mL insulin. (Humulin; Eli Lilly, IN, USA), 10 ng/mL human recombinant epidermal growth factor (Higeta Shoyu, Chiba, Japan), 1 nmol/L cholera toxin (Fujifilm Wako Pure Chemicals), 2 nmol/L triiodothyronine. (Fujifilm Wako Pure Chemicals) and Ham's F-12 nutrient medium (Thermo Fisher Scientific) supplemented with 0.4 mg/mL hydrocortisone (Saxizon; Teva Takeda Pharma, Aichi, Japan) in a 3:1 ratio. It becomes.

After approximately 2 weeks of culture, hESC-derived keratinocytes were stained with crystal violet, and the effective rate of colony formation was calculated by a conventionally known method (Reference 5, described below). Colonies of hESC-derived keratinocytes were classified into three groups: keratinocyte colonies, non-stratified epithelial colonies, and fibroblast-like colonies.

[5. Gene expression analysis of hESC-derived keratinocytes]
Gene expression analysis for keratin 14 (KRT14), tumor protein p63 (TP63), involucrin (IVL), and filaggrin (FLG) was performed using primer sequence data according to previous studies (see references below). Reference 6). Briefly, full-length RNA samples were prepared from 5th and 9th generation hESC-derived keratinocytes using the RNeasy Plus kit (Qiagen, Hilden, Germany) and transfected with SuperScript III Reverse Transcriptase (Thermo
Fisher Scientific) was used for cDNA synthesis.

mRNA expression was measured using Platinum SYBR Green qPCR SuperMix-UDG (Thermo Fisher Scientific) and Applied Biosystems.
Analysis was performed using Quantstudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific). The expression level was expressed as the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the mean ± SD of the ratio of GAPDH (%GAPDH) was shown.

Full-length RNA samples were prepared from third generation human postnatal epidermal keratinocytes and used as positive controls for detection of gene expression. Keratinocytes were co-cultured with a mouse feeder layer for 8 days in KCM supplemented with 10 mmol/L Y-27632. The cultured keratinocytes were cultured for 3 days in KCM supplemented with Y-27632 to induce terminal differentiation.

[6. Airlift culture method]
Keratinocytes derived from hESCs of the 16th generation (PDL31) were used to create a stratified epithelial equivalent by airlift culture according to a conventionally known method. Fifth generation postnatal epithelial keratinocytes were used as control cells. Briefly, mouse feeder layers were seeded onto LN-511-E8 coated 6-well cell culture inserts (Corning, NY, USA) at a density of 2.5×10 ⁴ cells/cm ² .

hESC-derived keratinocytes and postnatal epithelial keratinocytes were seeded onto the inserts at a density of 1 x ¹⁰⁴ cells/ ^cm2 and 0.5 x ¹⁰⁴ cells/ ^cm2 , respectively. These keratinocytes were cultured in KCM supplemented with 10 mmol/L Y-27632. After 7 days of culture, the culture medium in the culture insert was discarded, and airlift culture was performed in KCM not containing Y-27632. After 10 days of culture, keratinocytes were collected from the culture inserts as a layered epithelial equivalent and fixed with a 20% formalin solution.

[7. Histological analysis of epithelial equivalents]
Cultured hESC-derived keratinocytes and fixed epithelial equivalents of airlift cultured postnatal epidermis were processed into 5 mm thick paraffin wax embeddings according to standard methods and stained with eosin (HE) using standard methods. The expression of E-cadherin (CDH1), pankeratin (KRTs), KRT14, keratin (KRT10), IVL, TP63, and vimentin (VIM) was analyzed by immunohistological techniques. The implants were treated with mouse monoclonal anti-CDH1 (1:100, 36/E-Cadherin, Becton Dickinson, NJ, USA), mouse monoclonal anti-KRTs (1:100 dilution, AE1/AE3, Thermo Fisher Scientific), and mouse monoclonal anti-KRT14. (1:100, LL002, Abcam, Cambridge, UK), rabbit polyclonal anti-KRT10 (1:100, BioLegend, CA, USA), mouse monoclonal anti-IVL (1:1000, SY5, SigmaeAldrich), mouse monoclonal anti-TP63 (1 :100, 4A4, Abcam) and mouse monoclonal anti-VIM (1:100, Vim 3B4, Dako, Agilent Technologies, CA, USA) for 90 minutes at room temperature, followed by peroxidase-conjugated second antibody (Nichirei Biosciences). , Tokyo, Japan) according to the manufacturer's instructions. Human dermal and epithelial tissues were used as negative controls for immunohistological analysis.

[8. Statistical analysis]
A two-group t-test with Bonferroni correction was performed on the colony forming ability of keratinocytes, non-stratified epithelial cells, and fibroblast-like cells in the control group and the trypsin-treated group (n=6). Gene expression in hESC-derived keratinocytes and postnatal epithelial keratinocytes at the 5th and 9th generations was analyzed by a two-group t-test with Bonferroni correction (n=3). The density of hESC-derived cells was measured to confirm the effect of medium conditions and analyzed by two-group t-test with Bonferroni correction (n=3). Doubling times of hESC-derived keratinocytes obtained with or without Y-27632 were similarly analyzed by two-group t-test (n=3).

[9. result〕
(Separation of hESC-derived keratinocytes)
Prior to continuous culture, keratinocytes were isolated from hESC-derived differentiated cell populations using their sensitivity to trypsin. That is, before continuous culture, cells other than keratinocyte-like cells were removed by treating a cell group differentiated from hESC with trypsin-EDTA and removing the supernatant. After trypsin treatment, cells adhered to the culture dish were collected and subjected to continuous culture.

As shown in Figure 2, the ratio of keratinocytes in the trypsin-treated group improved as the continuous culture progressed compared to the control group. FIG. 2 is a diagram showing the cell morphology of a control group and a trypsin-treated group of hESC-derived keratinocytes at the 5th, 6th, and 7th generations. The left side of FIG. 2 shows the control group, and the right side shows the trypsin-treated group. In FIG. 2, the upper row shows the fifth generation (p5), the middle row shows the sixth generation (p6), and the lower row shows the seventh generation (p7). In FIG. 2, keratinocytes in the differentiated hESC cell population are indicated by dotted lines. The scale bar in FIG. 2 indicates 500 mm.

In the 6th generation (PDL2.0), cells recovered by the first trypsin treatment were cultured under continuous culture conditions and a conventional fibroblast culture method using α-MEM supplemented with FBS (Figure 3). . Since keratinocytes do not proliferate in conventional fibroblast medium, proliferating cells observed in α-MEM supplemented with FBS were classified as proliferating non-keratinocytes. FIG. 3 is a diagram showing the morphology of cells recovered by the first trypsin treatment in the 6th generation (PDL2.0). Cells were cultured under continuous culture conditions (left side in Figure 3). Cells were also seeded on standard culture dishes without a feeder layer and cultured in α-MEM medium supplemented with 10% fetal bovine serum (FBS) (10% FBS-α-MEM, Figure 3 center right). Under this culture condition, non-keratinocyte cells were observed in 10% FBS-α-MEM. The scale bar in FIG. 3 indicates 500 mm.

These results indicate that additional trypsin treatment is beneficial for the separation of keratinocytes from other differentiated hESCs.

In order to confirm the effect of trypsin treatment, the colony forming ability of the control group and trypsin-treated group was analyzed in the 7th generation. During culture, colonies classified into three types: keratinocytes, non-stratified epithelial, and fibroblast-like colonies were observed in both groups (A in FIG. 4). The effect of keratinocyte colony formation in the trypsin-treated group was significantly higher than that in the control group (B in Figure 4). Fibroblast-like cells were observed during continuous culture in the trypsin-treated group at the 7th generation (PDL3.7). (see Figure 2), but such colonies were not observed in either group (A in Figure 4).

FIG. 4 is a diagram showing the colony formation effect of hESC-derived keratinocytes. A in FIG. 4 is a diagram showing the morphology of hESC-derived keratinocytes in the control group (left side) and the trypsin-treated group (right side). Three types of colonies were observed in microscopic images and classified into keratinocyte (KC), non-stratified epithelial (NS), and fibroblast (FB)-like colonies. The scale bar in FIG. 4 indicates 500 mm. B in FIG. 4 shows the percentage of colony forming effect (%CFE) of hESC-derived keratinocytes in the control group and the trypsin-treated group. The percentage of colony forming effect was calculated for each of the three types of colonies, and compared between the control group and the trypsin-treated group. In B in FIG. 4, the white bar graph shows the results of the control group, and the black bar graph shows the results of the trypsin-treated group.

To confirm the presence of a proliferating non-keratinocyte cell population after further continuous culture, 13th generation (PDL23) hESC-derived keratinocytes were cultured at 10% concentration using standard culture dishes without a mouse feeder layer. The cells were cultured in α-MEM medium supplemented with FBS. Only hESC-derived keratinocytes were observed in continuous culture conditions, and no proliferating non-keratinocytes were observed in α-MEM (FIG. 5). FIG. 5 shows the morphology of 13th generation (PDL23) hESC-derived keratinocytes, with the left side showing the results under continuous culture conditions and the right side showing the results under fibroblast culture conditions. hESC-derived keratinocytes were also seeded in α-MEM medium supplemented with 10% FBS on standard culture dishes without a mouse feeder layer. In this culture condition, no proliferative non-keratinocyte cells were observed among the hESC-derived keratinocytes. The scale bar in FIG. 5 indicates 500 mm.

These results indicated that trypsin treatment is beneficial for the separation of keratinocytes from differentiated hESC cell populations. Moreover, the continuous culture conditions reliably reduced the proliferation of non-keratinocyte cells in hESC-derived keratinocytes.

(Gene expression of hESC-derived keratinocytes)
In order to prove the colony forming ability of hESC-derived keratinocytes, gene expression analysis was performed, and whether the hESC-derived keratinocytes contained keratinocyte progenitor cells was confirmed by qRT-PCR. Gene expression analysis was performed on the expression of KRT14, known as a marker for basal cells of epithelial tissue, and TP63, a marker for keratinocyte progenitor cells. Significantly higher expression was observed in the 9th generation hESC-derived keratinocytes than in the 5th generation (A in Figure 6).

FIG. 6 is a diagram showing the results of gene expression analysis of hESC-derived keratinocytes. Cultured human epithelial keratinocytes (KC) were used as a positive control for qRT-PCR. A in FIG. 6 shows the results of marker gene expression of keratinocyte precursor cells in 5th and 9th generation hESC-derived keratinocytes (p5 and p9, respectively). Gene expression of keratin 14 (KRT14) and tumor protein p63 (TP63) is shown as mean ± SD of percentage glyceraldehyde 3-phosphate dehydrogenase (%GAPDH). B in FIG. 6 shows marker gene expression for terminal differentiation of keratinocytes in 5th and 9th generation hESC-derived keratinocytes. Gene expression of involucrin (IVL) and filaggrin (FLG) is shown as mean ± SD of %GAPDH. In B in FIG. 6, the Y axis indicates logarithm.

Interestingly, there was no significant difference in gene expression of KRT14 and TP63 between 9th generation hESC-derived keratinocytes and 3rd generation cultured human epithelial keratinocytes. Signal gene expression of IVL and FGL keratinocyte terminal differentiation markers was significantly lower in 9th generation hESC-derived keratinocytes than in epithelial keratinocytes. The results of these gene expression analyzes showed that keratinocyte progenitor cells were enriched in hESC-derived keratinocytes by trypsinization and continuous culture, and that terminal differentiation of cells was achieved using serum-free low calcium ion medium and Y-27632. This shows that it is suppressed under continuous culture conditions.

(Stratified epithelial equivalent with hESC-derived keratinocytes)
hESC-derived keratinocytes were successfully isolated from the differentiated hESC cell population by additional trypsin treatment and continuous culture. In order to confirm the ability of hESC-derived keratinocytes to terminally differentiate into stratified epithelial keratinocytes, keratinocytes were seeded in cell culture inserts for airlift culture. Histological analysis of airlift cultures showed that hESC-derived keratinocytes formed a stratified epithelial equivalent with a stratum corneum, similar to postnatal keratinocytes (B in Figure 7). FIG. 7 shows the results of airlift culture of hESC-derived keratinocytes. A in FIG. 7 shows the morphology of hESC-derived keratinocytes after airlift culture, and B in FIG. 7 shows a paraffin-embedded piece of the equivalent stained with hematoxylin and eonin. The scale bar B in FIG. 7 indicates 200 mm.

Epithelial cell markers CDH1 and KRTs were expressed in hESC-derived keratinocytes and epithelial tissue, and mesenchymal cell marker VIM was not expressed (Figure 8). Furthermore, hESC-derived keratinocytes and epithelial-derived keratinocytes expressed not only progenitor cell markers KRT14 and TP63 but also terminal differentiation markers KRT10 and IVL (FIG. 8). FIG. 8 shows marker expression in epithelial equivalents derived from hESC-derived keratinocytes. Epithelial equivalents were generated by airlift culture. In Figure 8, postnatal human epithelial keratinocytes are shown on the left, and hESC-derived keratinocytes are shown on the right. The equivalents were analyzed by immunohistological methods to confirm the expression of E-cadherin (CDH1), pankeratin (KRTs), KRT14, keratin (KRT10), IVL, TP63, and vimentin (VIM). The scale bar in FIG. 8 indicates 200 mm.

The proportions of TP63-positive cells were 36.7±3.0 and 33.0±5.4 (mean±SD) in hESC-derived keratinocytes and postnatal epithelial keratinocytes, respectively. This indicates that there is no significant difference between the two types of keratinocytes. These results indicate that the differentiation potential of hESC-derived keratinocytes is maintained during long-term continuous culture under the conditions of this experiment.

(Effects of Y-27632 and LN-511-E8 on hESC-derived keratinocytes)
After isolation by trypsinization, hESC-derived keratinocytes were continuously cultured on culture dishes coated with LN-511-E8 with a mouse feeder layer in DK-SFM supplemented with Y-27632.

Figure 9 shows the results of continuous culture of hESC-derived keratinocytes, and shows the PDL of hESC-derived keratinocytes from the 5th generation (PDL0) to the 22nd generation (PDL45). The PDL of fifth generation keratinocytes before separation by trypsin treatment was defined as 0. The morphology of hESC-derived keratinocytes during continuous culture is shown in FIG. 9 for the 5th generation (PDL0), 11th generation (PDL16), 16th generation (PDL31), and 22nd generation (PDL45). The scale bar in FIG. 9 indicates 200 mm.

The doubling times of PDLs from the 5th generation (PDL0) to the 22nd generation (PDL45) and from the 7th generation (PDL3.7) to the 21st generation (PDL44) were calculated and shown in FIGS. 10 and 11. FIG. 10 shows the growth curve of hESC-derived keratinocytes in continuous culture by PDL from the 5th generation (PDL0) to the 22nd generation (PDL45). Figure 11 shows the doubling time of hESC-derived keratinocytes during continuous culture from the 7th generation (PDL3.7) to the 21st generation (PDL44).

The subcultured hESC-derived keratinocytes exhibited a logarithmic growth phase up to the 19th generation (PDL41), and the doubling time in the growth phase was 2.3±0.14 days (mean±SD). This result indicates that Y-27632 and LN-511-E8 may be essential components for promoting long-term culture.

(Study of continuous culture conditions)
The necessity of each component included in the continuous culture conditions was confirmed as shown below. Keratinocytes derived from hESC were cultured under the five culture conditions shown in Table 1 below.

Culture condition 1 is a continuous culture condition using LN-511-E8, Y-27632 and a feeder layer. Culture condition 2 is a condition in which Y-27632 is removed from culture condition 1. Culture condition 3 is a condition in which LN-511-E8 is removed from culture condition 1. Culture condition 4 is a condition in which LN-511-E8 and Y-27632 are removed from culture condition 1. Culture condition 5 is a condition obtained by removing the feeder layer from culture condition 1.

By evaluating five culture conditions of hESC-derived keratinocytes in terms of cell density and cell doubling time after culturing, the necessity of LN-511-E8, Y-27632 and feeder layers for maintaining cell proliferative properties was evaluated. It was confirmed. Microscopic images of hESC-derived keratinocytes cultured under each culture condition are shown in FIG. 12, and cell densities are shown in FIG. 13. FIG. 12 is a diagram showing the cell morphology of hESC-derived keratinocytes cultured under five culture conditions. The scale bar in FIG. 12 indicates 200 mm. Figure 13 shows the cell density of hESC-derived keratinocytes cultured under five culture conditions.

As shown in Figures 12 and 13, hESC-derived keratinocytes cultured under culture condition 1 had a significantly higher cell density than hESC-derived keratinocytes cultured under culture conditions 2 to 5.

Additionally, the results of comparing the doubling times between culture conditions 1 and 2 are shown in FIG. FIG. 14 shows the doubling time of hESC-derived keratinocytes cultured under culture conditions 1 and 2. As shown in FIG. 14, hESC-derived keratinocytes cultured under culture condition 1 had a significantly shorter doubling time than hESC-derived keratinocytes cultured under culture conditions 2 to 5.

These results showed that long-term continuous culture of hESC-derived keratinocytes was possible under continuous culture conditions including LN-511-E8, Y-27632, and a feeder layer.

(Study of trypsin treatment conditions)
Using already purified hESC-derived keratinocytes, the percentage of detached cells was measured for each trypsin treatment time. Keratinocytes derived from hESC were treated with 0.25 w/v% trypsin 1 mmol/L EDTA solution at 37°C. When the percentage of cells contained in the supernatant after treatment was measured, it was 5.63 ± 0.54% (n = 3) when the trypsin treatment time was 3 minutes, and 11/1 ± 0.82 when the trypsin treatment time was 6 minutes. % (n=3), and 60.9±8.76% (n=3) for 9 minutes.

On the other hand, in one aspect of the present invention, the cells removed by the first trypsin treatment are considered to be mainly fibroblasts based on their cell morphology. Therefore, hESC-derived fibroblasts were prepared and the number of cells recovered by trypsin treatment was compared with hESC-derived keratinocytes.

When hESC-derived fibroblasts were treated with a 0.25 w/v% trypsin 1 mmol/L EDTA solution at 37°C for 3 minutes, it was observed that all cells detached from the culture surface. On the other hand, when the same treatment was carried out for 1 minute, it was confirmed that many cells maintained their adhesion to the culture dish. When the number of cells recovered after trypsin treatment was measured, it was 14.1 ± 0.79 (x10 ⁵ cells/culture dish, n = 3) when trypsin treatment was for 3 minutes, and 5.3 ± when trypsin treatment was for 1 minute. It was 0.7 (x10 ⁵ pieces/culture dish). Regarding hESC-derived fibroblasts, it was confirmed that there was a significant difference in the amount of cells recovered depending on the treatment time with trypsin (t-test, p<0.01).

[References]
For references herein, see:
Reference 1: Akutsu H, Machida M, Kanzaki S, Sugawara T, Ohkura T, Nakamura N, et al. Xenogeneic-free defined conditions for derivation and expansion of human embryonic stem cells with mesenchymal stem cells. Regen Ther 2015;1: 18-29
Reference 2: Yachida S, Wood LD, Suzuki M, Takai E, Totoki Y, Kato M, et al. Genomic sequencing identifies ELF3 as a driver of ampullary carcinoma. Cancer Cell
2016;29:229-40
Reference 3: Takagi R, Yamato M, Kushida A, Nishida K, Okano T. Profiling of extracellular matrix and cadherin family gene expression in mouse feeder layer cells: type VI collagen is a candidate molecule inducing the colony formation of epithelial cells. Tissue Eng 2012;18:2539-48
Reference 4: Takagi R, Yamato M, Murakami D, Kondo M, Yang J, Ohki T, et al. Preparation of keratinocyte culture medium for the clinical applications of regenerative medicine. J Tissue Eng Regen Med 2011;5:e63-73 .
Reference 5: Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975;6:331-43.
Reference 6: Kajiwara K, Tanemoto T, Wada S, Karibe J, Ihara N, Ikemoto Y, et al. Fetal therapy model of myelomeningocele with three-dimensional skin using amniotic fluid cell-derived induced pluripotent stem cells. Stem Cell Rep 2017 ;8:1701-13.

The present invention can be used in the medical field.

Claims (11)

A method for producing keratinocytes in which a cell population containing keratinocytes derived from pluripotent stem cells is subjected to multiple separation steps of separating keratinocytes by treating with trypsin. The method for producing keratinocytes according to claim 1, wherein in the separation step, keratinocytes are separated based on differences in cell adhesiveness after trypsin treatment. In the separation step,
Performing a first trypsin treatment to detach cells other than keratinocytes from the container on a cell population including keratinocytes adhered to the container,
After the first trypsin treatment, the cell population adhered to the container is subjected to a second trypsin treatment to detach the keratinocytes from the container, thereby separating the keratinocytes.
A method for producing keratinocytes according to claim 2. In the first trypsin treatment, a cell population containing keratinocytes is exposed to trypsin for 1 minute or more and 3 minutes or less,
4. The method for producing keratinocytes according to claim 3, wherein in the second trypsin treatment, the cell population from which detached cells have been removed by the first trypsin treatment is exposed to trypsin for 3 minutes or more. A method for producing keratinocytes, which includes a culture step of continuously culturing keratinocytes derived from pluripotent stem cells in the presence of feeder cells, a Rho kinase inhibitor, and an extracellular matrix. The method for producing keratinocytes according to claim 5, wherein the Rho kinase inhibitor is Y27632. The method for producing keratinocytes according to claim 5, wherein the extracellular matrix is laminin or a fragment thereof. A cell population containing keratinocytes derived from pluripotent stem cells is subjected to multiple isolation steps in which keratinocytes are separated by treatment with trypsin.
A method for producing keratinocytes, which comprises continuously culturing separated keratinocytes in the presence of feeder cells, a Rho kinase inhibitor, and an extracellular matrix. A medium kit for continuous culture of keratinocytes derived from pluripotent stem cells, including feeder cells, a Rho kinase inhibitor, and an extracellular matrix. Keratinocytes derived from pluripotent stem cells and having a population doubling level (PDL) of more than 15. The keratinocytes according to claim 10, wherein the cell population doubling time is 60 hours or less.

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