CN112980775B

CN112980775B - Culture solution for preparing keratinocyte based on differentiation of pluripotent stem cells

Info

Publication number: CN112980775B
Application number: CN202110297373.XA
Authority: CN
Inventors: 高歌; 周安宇
Original assignee: Ixcell Biotechnology Co ltd
Current assignee: Ixcell Biotechnology Co ltd
Priority date: 2021-03-19
Filing date: 2021-03-19
Publication date: 2024-01-23
Anticipated expiration: 2041-03-19
Also published as: CN112980775A

Abstract

The invention discloses a culture solution for preparing keratinocytes based on multipotent stem cell differentiation, which comprises a first-stage culture solution and a second-stage culture solution, wherein the first-stage culture solution comprises the following components: DMEM/F12 basal broth, KOSR, optional amino acids NEAA, L-Glutamine, β -mercaptoethanol, SU6656, retinoic acid, CHIR99021, hEGF, and NKH477; the second stage culture solution comprises: DMEM/F12 basal broth, KOSR, optional amino acids NEAA, L-Glutamine, β -mercaptoethanol, BMP4 and ascorbic acid. The culture solution can obtain mature endothelial cells from induced multifunctional stem cells in a short time, and the primary keratinocytes and the mature keratinocytes obtained by the method have good purity, high activity rate and complete functions, and have potential clinical application value.

Description

Culture solution for preparing keratinocyte based on differentiation of pluripotent stem cells

Technical Field

The invention belongs to the technical field of cell engineering, and particularly relates to a culture solution for preparing keratinocytes based on differentiation of pluripotent stem cells.

Background

At present, the skin is used as a largest human organ and is also a first barrier of a human body, and has various functions of preventing invasion of pathogenic bacteria, preventing body dehydration, regulating body temperature, providing perception and the like. Keratinocytes are located in the basal layer of the deepest stratified epithelium, sometimes referred to as basal cells or basal keratinocytes. It is known that 95% of cells in epidermis are keratinocytes, squamous keratinocytes are also present in mucous membranes of the oral cavity and esophagus, and cornea, conjunctiva, genital epithelial cells and the like are also keratinocytes. Keratinocytes maintain various stages of epidermal differentiation and are responsible for forming Tight junctions with the nerves of the skin (light junctions). They also support Langerhans cells (Langerhans cells) of dermis, epidermis and lymphocytes.

Keratinocytes play a role in the immune system function in addition to their structural role. Skin acts as a first line of defense, keratinocytes being a barrier between an organism and its environment. In addition to preventing toxins and pathogens from entering and exiting the body, they also prevent the loss of body moisture, heat and other important components. Meanwhile, it is responsible for secreting inhibitory cytokines without injury, stimulating inflammatory stress, and activating Langerhans cells (Langerhans cells) response to injury. Langerhans cells, as antigen presenting cells, are the first cells to process microbial antigens from the site of skin rupture into the body when the skin is infected.

In organisms, keratinocytes are replaced and supplemented by stem cells in the basal layer of the epidermis. The compounds and other proteins produced by keratinocytes are critical to the integrity of the outermost stratum corneum of the skin, but keratinocytes are dead squamous cells and have no reproductive capacity. Once the keratinocytes reach the stratum corneum, they become keratinized, forming a tough skin, the stratum corneum, which is thought to have a major role in defending against microbial invasion, and also act as an activator of immune response.

A population of cells with antigen presenting effects in the epidermis, capable of activating T lymphocytes under appropriate conditions to accommodate cellular and humoral immune responses. Of these, langerhans cells play a major role, but keratinocytes also play a role under specific conditions.

Currently, keratinocytes have been available as skin implants, either alone or in combination with other ingredients. Therefore, it is widely used for cell therapy in view of the role of keratinocytes in forming a physical barrier and the ability to produce antibacterial peptides. Extensive burns and diseases such as diabetes, vascular disease, obesity, chronic wound injury have become a global challenge due to lack of effective treatment, poor treatment efficacy, and huge costs. Cell transplantation therapy is currently the most timely and effective treatment for the above problems, and is usually performed by in vitro proliferation of the patient's own keratinocytes stem cells to obtain a sufficient amount of cells. However, the greatest disadvantage of this approach is that obtaining sufficient stem cells generally requires a long period, which may lead to dehydration and infection of the patient's body. In addition, there is a problem in that immune rejection is involved, and the transplanted epidermal cells may be rapidly rejected.

In order to overcome such problems, early researchers tried to propose the use of inert or biosynthetic matrices, but these were abandoned due to rapid graft rejection and to diseases caused by the inclusion of heterologous collagens and adult heterologous skin cells. Pluripotent stem cells, including embryonic stem cells (embryonic stemcells, ESCs) and induced pluripotent stem cells (induced Plurpotent Stem Cells, iPSCs), hold promise for this therapeutic modality. Because pluripotent stem cells can proliferate indefinitely and have multipotency that can differentiate into any cell type, they are widely used in the development of clinically useful cell therapy products. Although embryonic stem cells have been reported to differentiate into keratinocytes, they have low proliferation potential and cannot be replicated on a large scale. Later, some methods of short-term induction of cytokines were also used to differentiate hESCs into keratinocytes. However, there is still some difficulty in producing functional basal keratinocytes. Furthermore, in view of the ethical advantages of hiPSCs over hESCs, clinical application and transformation of keratinocytes derived from iPSCs is more significant.

Disclosure of Invention

1. Technical problem to be solved by the invention

The invention aims to solve the technical problems of long preparation period, low yield and purity, incomplete cell function and the like in the existing method for inducing and differentiating keratinocytes by using pluripotent stem cells.

2. Technical proposal

In order to achieve the above purpose, the technical scheme provided by the invention is as follows:

the invention discloses a culture solution for preparing keratinocytes based on multipotent stem cell differentiation, which comprises a first-stage culture solution and a second-stage culture solution, wherein the first-stage culture solution comprises the following components: DMEM/F12 basal broth, KOSR, optional amino acids NEAA, L-Glutamine, β -mercaptoethanol, SU6656, retinoic acid, CHIR99021, hEGF, and NKH477; the second stage culture solution comprises: DMEM/F12 basal broth, KOSR, optional amino acids NEAA, L-Glutamine, β -mercaptoethanol, BMP4 and ascorbic acid.

Preferably, the volume ratio of DMEM/F12 basal culture solution in the first stage culture solution and the second stage culture solution is 1:0.9-1.3, the concentration of KOSR in the first stage culture solution and the second stage culture solution is 15% -30%, the concentration of NEAA (non-essential amino acid) in the first stage culture solution and the second stage culture solution is 1%, the concentration of L-Glutamine in the first stage culture solution and the second stage culture solution is 0.5-1mM, and the concentration of beta-mercaptoethanol in the first stage culture solution and the second stage culture solution is 0.05-1.20mM.

Preferably, the concentration of SU6656 in the culture solution of the first stage is 3-10 mu M, the concentration of Retinoic Acid (RA) is 0.5-2 mu M, CHIR and 99021 is 5-20mM, the concentration of hEGF is 8-16ng/mL, and the concentration of NKH477 is 0.05-0.2mM.

Preferably, the BMP4 concentration in the second stage culture solution is 0.05-0.8nM and the ascorbic acid concentration is 0.05-2. Mu.M.

Preferably, the volume ratio of the DMEM/F12 basal medium is 1:1, the concentration of KOSR is 20%, the concentration of L-Glutamine is 1mM, and the concentration of beta-mercaptoethanol is 0.1mM.

Preferably, the SU6656 is 6. Mu.M, retinoic Acid (RA) is 1. Mu. M, CHIR 99021:99021 is 10mM, hEGF is 10ng/mL, and NKH477 is 0.1mM.

Preferably, the BMP4 concentration is 0.6nM and the ascorbic acid concentration is 1. Mu.M.

3. Advantageous effects

Compared with the prior art, the technical scheme provided by the invention has the following beneficial effects:

the invention discloses a culture solution for preparing keratinocytes based on multipotent stem cell differentiation, which comprises a first-stage culture solution and a second-stage culture solution, wherein the first-stage culture solution comprises the following components: DMEM/F12 basal broth, KOSR, optional amino acids NEAA, L-Glutamine, β -mercaptoethanol, SU6656, retinoic acid, CHIR99021, hEGF, and NKH477; the second stage culture solution comprises: DMEM/F12 basal broth, KOSR, optional amino acids NEAA, L-Glutamine, β -mercaptoethanol, BMP4 and ascorbic acid. The culture solution of the invention can obtain mature endothelial cells from induced multifunctional stem cells in a short time, and the primary keratinocytes and the mature keratinocytes have good purity, high activity rate and complete functions, and have potential clinical application value.

Drawings

FIG. 1 is a flow chart of keratinocyte production in accordance with the present invention;

FIG. 2 is a schematic diagram showing the results of flow cytometry detection of surface marker (K18) in example 2;

FIG. 3 is a schematic diagram showing the results of flow cytometry detection of surface marker (K14) in example 3;

FIG. 4 is a schematic representation of a human induced pluripotent stem cell pattern according to the invention;

FIG. 5 is a schematic representation of the primary keratinocyte morphology according to the present invention;

FIG. 6 is a schematic representation of the mature keratinocytes of the present invention;

FIG. 7 is a schematic representation of the results of primary keratinocyte flow cytometry detection of surface markers (K18, p 63) of examples 4-7;

FIG. 8 is a schematic representation of the results of detection of surface markers (K14) by flow cytometry on mature keratinocytes of examples 8-11.

Detailed Description

In order that the invention may be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which, however, the invention may be embodied in many different forms and are not limited to the embodiments described herein, but are instead provided for the purpose of providing a more thorough and complete disclosure of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; the terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention; the term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

Example 1

The culture solution for preparing the keratinocytes based on the differentiation of the pluripotent stem cells comprises a first-stage culture solution and a second-stage culture solution, wherein the first-stage culture solution comprises the following components: DMEM/F12 basal broth, KOSR, optional amino acids NEAA, L-Glutamine, β -mercaptoethanol, SU6656, retinoic acid, CHIR99021, hEGF, and NKH477; the second stage culture solution comprises: DMEM/F12 basal broth, KOSR, optional amino acids NEAA, L-Glutamine, β -mercaptoethanol, BMP4 and ascorbic acid.

Wherein the specific components of the first-stage culture solution are as follows:

wherein the specific components of the second stage culture solution are shown in the following table:

reagent(s)	Preferred concentration	Suppliers (suppliers)
			DMEM/F12	1：1	Life Technologies，USA
KOSR	20％	Life Technologies，USA
			Optional amino acid NEAA	1×	Life Technologies，USA
L-Glutamine	1mM	Life Technologies，USA
			Beta-mercaptoethanol	0.1mM	Sigma，USA
BMP4	0.6nM	R&D system，UK
			Ascorbic acid	1μM	Sigma，USA

The method of using the culture solution of this example was as follows:

after in vitro culture of Induced Pluripotent Stem Cells (iPSCs) to 10 passages, cells with a degree of confluence of 70% in cell culture plates were cultured, digested into single cells with Accutase (STEMCELL Technologies), plated in 12-well plates pre-coated with Martrigel (BD) at a density of (30000 cells/cm 2), and ROCK inhibitor (10 μm) (Day 0) was added.

After 24 hours (Day 1), the primary keratinocyte differentiation was performed by changing to 1mL/Well first stage culture medium, changing to every 2 days, culturing by changing to 1mL/Well KGM medium when the cells grew to 70% (about Day 6), changing to every Day until the cells grew to 80% -90% confluence (about Day12, day 12), the cells exhibited keratinocyte morphology, and collecting a portion of the cells for flow detection, thereby obtaining the primary keratinocyte expressing Cytokeratin18 (K18) marker.

Primary keratinocytes were passaged after digestion, re-plated on fresh Matrigel coated plates at a ratio of 1:3, replaced daily by keratinocyte proliferation medium (KEM), and primary keratinocytes could proliferate in this case for more than 10 passages and continue to express K18 (efficiency > 99%).

Continuing to differentiate into mature keratinocytes

The primary keratinocytes expressing the K18 marker are subjected to continuous differentiation to obtain mature keratinocytes expressing K14.

The method comprises the following steps:

(1) When the primary keratinocytes obtained above were cultured until the polymerization degree became 50%, the medium was changed to the second-stage culture medium, and 1.5mL/Well of the second-stage culture medium was changed every day for 6 days.

(2) On day 7 the medium was changed to KGM medium at 2 mL/Well. (K14 is a marker for many cells of basal layer of epidermal tissue and dermal tissue). In addition, the transcription factor p63 regulates the regeneration capacity of epidermal tissues, and is expressed in the whole process of epidermal differentiation. And collecting part of cells for flow detection to obtain mature keratinocytes with strong expression of K14.

The advantages of using the method of the invention are: the method has the advantages of simple operation and high efficiency, can obtain a large number of primary keratinocytes in a short time (about 12 days), and has uniform keratinocyte morphology and cobblestone shape of typical keratinocytes.

The primary keratinocyte and the mature keratinocyte obtained by culture can be stored at ultralow temperature (liquid nitrogen) or used for basic research and even clinical use after cell type identification confirmation by flow cytometry (using keratinocyte characteristic markers).

The invention has the positive progress effects that:

firstly, the culture solution has simple components, is convenient to prepare, does not relate to animal-derived components, can control and reduce the cost, and ensures the safety of differentiated keratinocytes.

Secondly, the culture method is efficient and reliable, and the keratinocytes with high yield and high purity can be obtained through the improved culture solution.

Moreover, the culture method of the invention has obvious advantages compared with other methods, and is particularly shown in the following steps: the culture time is short (about 21 days), and the clinic use timeliness is achieved; the culture process is simple, and can be completed only by replacing two differentiation culture mediums; the culture efficiency is high, and the purity of the cells is over 95 percent; the induction process is clear and transparent, and the induction product has high purity.

Example 2

In this example, to test how different concentrations of SU6656, retinoid acid, CHIR99021, hEGF and NKH477 in the first stage culture solution affect the induction of primary keratinocytes, the basic components are the same as those of the first stage culture solution in example 1, except that different concentrations of SU6656, retinoid acid, CHIR99021, hEGF and NKH477 are used in this example to test what effect the induction of primary keratinocytes occurs at different concentrations, the specific contents and processes are as follows:

SU6656 control test component content table of different concentrations

Retinoic acid control test component content table with different concentrations

CHIR99021 control test component content table with different concentrations

hEGF control assay component content tables at different concentrations

NKH477 control test component content table of different concentrations

The total 25 solutions of the above 5 groups were used as follows:

1) inoculation of iPSCs

PBMC-derived 20 th generation human Induced Pluripotent Stem Cells (iPSCs) were digested into single cells, centrifuged, and suspended in mTeSR-1 medium supplemented with ROCK inhibitor (10 μm) and seeded into Matrigel pre-coated 12-well plates (12 wells) at a density of 30000 cells/cm 2. Culturing in an incubator with 5% CO2 at 37℃for 24 hours.

2) Induction of primary keratinocytes by varying concentrations of an inducer

The 12-Well plate after culturing for 24 hours in the step (1) was taken out, the medium was aspirated, and fresh 1mL/Well basal medium (basal medium of DMEM/F12, KOSR, optional amino acids NEAA, L-Glutamine, beta-mercaptoethanol, etc.) was replaced, and 25 kinds of the above solutions were added in total. Every two days, when the cells grow to the convergence degree of about 70% (about day 6), the cells are changed into 1mL/Well KGM culture medium, the liquid is changed every day until the cells grow to the convergence degree of 80% -90% (about day 12), the cells are in the form of keratinocytes, and part of the cells are collected for flow detection, wherein the detection result is shown in figure 2.

As is clear from FIG. 2, SU6656 at a concentration of 6. Mu.M gave the best effect of inducing primary keratinocytes, retinoic acid at a concentration of 1. Mu.M gave the best effect of inducing primary keratinocytes at a concentration of CHIR99021 at a concentration of 10mM, hEGF at a concentration of 12ng/mL gave the best effect of inducing primary keratinocytes, and NKH477 at a concentration of 0.1mM gave the best effect of inducing primary keratinocytes.

The primary keratinocytes obtained in example 2 were subjected to the characterization of the expression and purity of the characteristic protein (K18) by the following method:

1) Fixing cells

Primary keratinocytes cultured in a 12-well plate are rinsed once by using 1 xDPBS (0.5 mL/well), then 0.5-1 mL of Actutase digestive juice is added, and the primary keratinocytes are digested for 3-5 minutes at 37 ℃ until the cells are separated; then adding 2-4 times of DPBS to dilute the digestion liquid, centrifuging 200-300 g and discarding the digestion liquid and the DPBS. Cell pellet was fixed using 1mL of 90% cold methanol.

2) Incubation with primary antibody

Cell samples were centrifuged at 300rcf for 2 min at room temperatureThe supernatant was discarded. 5mL of 1 XPBS was added to the cell samples and mixed with shaking. Centrifuging at 300rcf for 3 min, removing supernatant, adding FACS buffer, and blow mixing to give cell concentration of 2×10 ⁷ And each mL. To the flow cytometer dedicated tube, 50 μl of the corresponding sample cells were added, respectively. 50 mu L of the corresponding primary antibody is added into the sample cells, the mixture is blown for 5 times and is incubated for 30 minutes at room temperature in a dark place (shaking and mixing are carried out every 10 minutes). To each of the cells after antibody incubation, 2mL of FACS buffer was added and mixed with shaking using a vortex mixer. The species and volumes of the primary antibodies are shown in the table below.

Antibody species and volume

3) Second antibody incubation

Centrifuge at 300rcf for 2 min at room temperature. The supernatant was discarded, 50. Mu.L of the corresponding secondary antibody was added to the sample cells, and the mixture was blown 5 times and incubated at room temperature for 15 minutes in the dark (the corresponding secondary antibody was selected according to the source of the primary antibody). mu.L of FACS buffer was added to each, and the mixture was homogenized by shaking using a vortex mixer. The corresponding flow cytometer channel was selected to detect the positive rate of each marker.

FIG. 2 is a graph showing the results of flow cytometry on K18 of primary cells obtained in example 2, wherein when SU6656, retinoic Acid (RA), CHIR99021, hEGF and NKH477 were added at a certain concentration, primary keratinocytes were obtained, and the expression level of characteristic protein K18 was started to be expressed, whereas the control group did not express K18. When the concentration of SU6656 is 3-10. Mu.M, preferably 6. Mu.M; retinoic Acid (RA) concentration is 0.5-2 μm, preferably 1 μm; CHIR99021 concentration is 5-20mM, preferably 10mM; hEGF concentration of 8-16ng/mL, preferably 12ng/mL and NKH477 concentration of 0.05-0.2mM, preferably 0.1 mM) the expression level of the characteristic protein K18 of the primary keratinocytes obtained was optimal.

Example 3

In this example, on the basis of example 2, the optimal concentrations of SU6656, retinoic acid, CHIR99021, hEGF and NKH477 were selected, primary keratinocytes were obtained by culturing in the manner of example 2, and the primary keratinocytes were replaced with fresh 1mL/Well basal medium (basal medium of DMEM/F12, KOSR, unnecessary amino acids NEAA, L-Glutamine, β -mercaptoethanol, etc.), and different concentrations of mature keratinocyte differentiation-specific components such as BMP4 and ascorbic acid were added, and the effect of different concentrations of BMP4 and ascorbic acid on the mature keratinocyte differentiation-specific components was tested.

The first stage broth used in this example had the following composition:

the second stage culture solution used in this example was tested for the effect of BMP4 and ascorbic acid on the induction of mature keratinocytes using different concentrations of BMP4 and ascorbic acid, and the specific contents and processes were as follows:

BMP4 control assay component content table for different concentrations

Sequence number/composition	BMP4	Ascorbic acid
			1	0nM	0.5μM
2	0.05nM	0.5μM
			3	0.2nM	0.5μM
4	0.4nM	0.5μM
			5	0.6nM	0.5μM
6	0.8nM	0.5μM

BMP4 control assay component content table for different concentrations

Sequence number/composition	BMP4	Ascorbic acid
			1	0.4nM	0μM
2	0.4nM	0.05μM
			3	0.4nM	0.5μM
4	0.4nM	1μM
			5	0.4nM	1.5μM
6	0.4nM	2μM

The 2 groups of 12 kinds of culture solutions were each cultured as follows:

on the basis of example 2, the primary keratinocytes induced by the most preferred concentration of the inducer were replaced with fresh 1mL/Well basal medium (basal medium of DMEM/F12, KOSR, optional amino acids NEAA, L-Glutamine, beta-mercaptoethanol, etc.), and mature keratinocyte differentiation-specific components of BMP4 and ascorbic acid, etc. were added at different concentrations. When the primary keratinocytes in the step (2) of example 2 were cultured until the polymerization degree was 50%, the culture medium was changed to a mature keratinocyte differentiation medium having different concentrations of the inducer, and the culture was continued for 6 days with 1.5mL/Well medium each day. On day 7 the medium was changed to KGM medium at 2 mL/Well.

As shown in FIG. 3, the results of flow-through measurement of the harvested cells of the mature keratinocytes obtained in example 3 show that the effect of inducing mature keratinocytes was optimal when the BMP4 concentration was 0.6nM and the effect of inducing mature keratinocytes when the ascorbic acid concentration was 1. Mu.M.

The mature keratinocytes obtained in example 3 were characterized for the expression and purity of the characteristic protein (K14) by the following method:

1) Fixing cells

Mature keratinocytes cultured in a 12-well plate are rinsed once by using 1 xDPBS (0.5 mL/well), then 0.5-1 mL of Actutase digestive juice is added, and the mixture is digested for 3-5 minutes at 37 ℃ until the cells are separated; then adding 2-4 times of DPBS to dilute the digestion liquid, centrifuging 200-300 g and discarding the digestion liquid and the DPBS. Cell pellet was fixed using 1mL of 90% cold methanol.

2) Incubation with primary antibody

The cell samples were centrifuged at 300rcf for 2 min at room temperature and the supernatant was discarded. 5mL of 1 XPBS was added to the cell samples and mixed with shaking. Centrifuging at 300rcf for 3 min, removing supernatant, adding FACS buffer, and blow mixing to give cell concentration of 2×10 ⁷ And each mL. To the flow cytometer dedicated tube, 50 μl of the corresponding sample cells were added, respectively. 50 mu L of the corresponding primary antibody is added into the sample cells, the mixture is blown for 5 times and is incubated for 30 minutes at room temperature in a dark place (shaking and mixing are carried out every 10 minutes). To each of the cells after antibody incubation, 2mL of FACS buffer was added and mixed with shaking using a vortex mixer. The species and volumes of the primary antibodies are shown in the table below.

Antibody species and volume

3) Second antibody incubation

FIG. 3 is a graph showing the results of flow cytometry on K14 of primary cells obtained in example 3, wherein the concentration of BMP4 in example 2 is 0.05-0.8nM, preferably 0.6nM; and the optimal expression level of the characteristic protein K14 of the mature keratinocytes obtained at an ascorbic acid concentration of 0.05 to 2. Mu.M, preferably 1. Mu.M.

Examples 4 to 7

Examples 4-7 specifically repeat the following 4 times:

(1) inoculation of iPSCs

(2) Induction of primary keratinocytes

On the basis of example 2, a preferred concentration inducer was selected, the 12-Well plate after 24 hours of incubation in step (1) was removed, the medium was aspirated off, and fresh 1mL/Well of the first-stage broth was replaced. Every two days, when the cells grow to the aggregation degree of about 70% (about day 6), changing to 1mL/Well KGM culture medium, changing the liquid every day until the cells grow to the aggregation degree of 80% -90% (about day 12), and collecting part of the cells for flow detection.

The primary keratinocytes obtained in examples 4 to 7 were each subjected to the characterization of the expression and purity of the characteristic proteins (p 63, K18) as follows:

1) Fixing cells

2) Incubation with primary antibody

The cell samples were centrifuged at 300rcf for 2 min at room temperature and the supernatant was discarded. 5mL of 1 XPBS was added to the cell samples and mixed with shaking. Centrifuging at 300rcf for 3 min, removing supernatant, adding FACS buffer, and blow mixing to give cell concentration of 2×10 ⁷ And each mL. To the flow cytometer dedicated tube, 50 μl of the corresponding sample cells were added, respectively. 50 mu L of the corresponding primary antibody is added into the sample cells, the mixture is blown for 5 times and is incubated for 30 minutes at room temperature in a dark place (shaking and mixing are carried out every 10 minutes). To each of the cells after antibody incubation, 2mL of FACS buffer was added and mixed with shaking using a vortex mixer. Primary antibodyThe types and volumes are shown in the following table.

Antibody species and volume

3) Second antibody incubation

FIG. 7 is a graph showing the results of flow cytometry identification of K18 and p63 of the primary cells obtained in examples 4-7, wherein the primary keratinocytes obtained in examples 4-7 have a characteristic protein K18 and p63 expression level of 99% or more; it can be demonstrated that the keratinocytes obtained by the culture solution and the culture method of the present invention grow homogeneously and efficiently.

Examples 8 to 11

The primary keratinocytes obtained in examples 4 to 7 were subjected to the following procedures to obtain examples 8 to 11, respectively

On the basis of example 3, a preferred concentration of inducer is selected and the primary keratinocytes of examples 3-6 are transferred to a mature keratinocyte medium for differentiation culture of the mature keratinocytes.

When the primary keratinocytes in step (2) of step examples 3 to 6 were cultured until the polymerization degree was 50%, the medium was changed to the second-stage culture medium, and 1.5mL/Well of the second-stage culture medium was changed every day for 6 days. On day 7, the second stage broth was changed to KGM medium at 2 mL/Well.

The mature keratinocytes obtained in examples 8-11 were subjected to characterization of the expression and purity of the characteristic protein (K14). The method comprises the following steps:

1) Fixing cells

2) Incubation with primary antibody

Antibody species and volume

3) Second antibody incubation

FIG. 8 is a graph showing the results of flow cytometry identification of K14 from primary cells obtained in examples 8-11, wherein the expression level of K14, characteristic of mature keratinocytes obtained in examples 8-11, was 95% or more; it can be demonstrated that the keratinocytes obtained by the culture solution and the culture method of the present invention grow homogeneously and efficiently.

The data from the above examples show that: by using the culture solution and the differentiation method disclosed by the invention, the mature endothelial cells derived from the induced multifunctional stem cells can be obtained in a short time, and the primary keratinocytes and the mature keratinocytes have the advantages of good purity, high activity rate, complete functions and potential clinical application value.

In examples 4-11, the plates were observed under a microscope and photographed before plating of primary keratinocytes differentiation (D0), and after induced differentiation (D6) and completion of differentiation of mature keratinocytes (D21).

The results are shown in fig. 4, 5 and 6: FIG. 4 is a diagram showing the cell morphology of the primary keratinocytes obtained by culturing the induced pluripotent stem cells having typical iPSC cell morphology, and FIG. 5 is a diagram showing the cell morphology of the primary keratinocytes obtained by culturing the cells of examples 4 to 7; FIG. 6 is a cell morphology of mature keratinocytes cultured in examples 8 to 11.

The foregoing examples merely illustrate certain embodiments of the invention and are described in more detail and are not to be construed as limiting the scope of the invention; it should be noted that it is possible for a person skilled in the art to make several variants and modifications without departing from the concept of the invention, all of which fall within the scope of protection of the invention; accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A culture solution for preparing keratinocytes based on multipotential stem cell differentiation is characterized in that: the method comprises a first-stage culture solution and a second-stage culture solution, wherein the first-stage culture solution is as follows: DMEM/F12 basal broth, KOSR, optional amino acids NEAA, L-Glutamine, β -mercaptoethanol, SU6656, retinoic acid, CHIR99021, hEGF, and NKH477; the second stage culture solution is as follows: DMEM/F12 basal broth, KOSR, optional amino acids NEAA, L-Glutamine, β -mercaptoethanol, BMP4 and ascorbic acid; placing the induced pluripotent stem cells in a first-stage culture solution for differentiation to obtain primary keratinocytes, and placing the primary keratinocytes in a second-stage culture solution for differentiation to obtain mature keratinocytes;

the volume ratio of DMEM/F12 basal culture solution in the first-stage culture solution and the second-stage culture solution is 1:0.9-1.3, the concentration of KOSR in the first-stage culture solution and the second-stage culture solution is 15% -30%, the concentration of NEAA (non-essential amino acid) in the first-stage culture solution and the second-stage culture solution is 1%, the concentration of L-Glutamine in the first-stage culture solution and the second-stage culture solution is 0.5-1mM, and the concentration of beta-mercaptoethanol in the first-stage culture solution and the second-stage culture solution is 0.05-1.20 mM;

the concentration of SU6656 in the culture solution of the first stage is 3-10 mu M, the concentration of Retinoic acid is 0.5-2 mu M, CHIR and 99021 is 5-20mM, the concentration of hEGF is 8-16ng/mL, and the concentration of NKH477 is 0.05-0.2mM;

the concentration of BMP4 in the second-stage culture solution is 0.05-0.8nM, and the concentration of ascorbic acid is 0.05-2. Mu.M.

2. The culture medium for preparing keratinocytes based on differentiation of pluripotent stem cells according to claim 1, wherein: the volume ratio of the DMEM/F12 basal culture solution is 1:1, the concentration of KOSR is 20%, the concentration of L-Glutamine is 1mM, and the concentration of beta-mercaptoethanol is 0.1mM.

3. The culture medium for preparing keratinocytes based on differentiation of pluripotent stem cells according to claim 1, wherein: the SU6656 concentration was 6 μm, retinoic acid concentration was 1 μ M, CHIR99021 concentration was 10mM, hEGF concentration was 12ng/mL, NKH477 concentration was 0.1mM.

4. The culture medium for preparing keratinocytes based on differentiation of pluripotent stem cells according to claim 1, wherein: the BMP4 concentration was 0.6nM and the ascorbic acid concentration was 1. Mu.M.