Culture method of induced pluripotent stem cells, kit and application

Abstract

The invention provides a culture method of induced pluripotent stem cells, a kit and application, and relates to the technical field of biology. The culture method of the induced pluripotent stem cells comprises the step of enabling the induced pluripotent stem cells to be attached to a cell culture medium for growth, wherein the cell culture medium contains exosomes. According to the culture method, the passage time of the induced pluripotent stem cells can be shortened, the damage of the induced pluripotent stem cells is reduced, and the number of the induced pluripotent stem cells is increased.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a culture method, a kit and an application of induced pluripotent stem cells. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Induced Pluripotent Stem Cells (iPSCs) are a type of cell with properties similar to those of embryonic stem cells. iPSCs reprogram somatic cells into stem cells by introducing specific transcription factors. The discovery of iPSCs is an important breakthrough in the field of stem cell research. It was initially achieved by transferring a combination of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) into differentiated somatic cells. iPSCs have properties similar to those of embryonic stem cells and can differentiate into multiple types of cells. This technology not only avoids the use of embryonic cells or oocytes, thereby solving related ethical issues, but has also attracted much attention for its potential applications in regenerative medicine, disease modeling, drug discovery, and other fields.

[0004] At present, the common culture method of iPSCs is to attach and grow iPSCs by laying matrix gel or VTN protein (Vitronectin) in the culture dish. After the somatic cells form clones, it is necessary to use a pipette to pick the clones under a microscope, and the cells need to be treated with digestive enzymes or EDTA during the passaging process. However, this method usually takes a long time to digest, which has a great impact on cell viability and cell yield. Therefore, improving the culture method of iPSCs is a problem that needs to be solved at present.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a method for culturing induced pluripotent stem cells to alleviate at least one defect of the above-mentioned induced pluripotent stem cells. Based on the method for culturing induced pluripotent stem cells, a kit for culturing induced pluripotent stem cells and their applications are also provided.

[0007] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0008] In a first aspect, a method for culturing induced pluripotent stem cells is provided, the method comprising allowing the induced pluripotent stem cells to adhere to a cell culture matrix and grow, wherein the cell culture matrix contains exosomes.

[0009] In a second aspect, a kit for inducing pluripotent stem cell culture is provided, the kit comprising a thermosensitive hydrogel, exosomes, and a culture medium for inducing pluripotent stem cells.

[0010] In a third aspect, there is provided a method for culturing induced pluripotent stem cells as described in the first aspect, or a kit for culturing induced pluripotent stem cells as described in the second aspect, and use thereof in the preparation of a cell medicine.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The induced pluripotent stem cell culture method provided by the present invention allows induced pluripotent stem cells to grow on a cell culture matrix containing exosomes, and the biological activity of exosomes is advantageously introduced into the cell culture system. Exosomes, as important carriers of intercellular communication, carry abundant bioactive molecules such as proteins and RNA, and can effectively simulate the in vivo microenvironment, promote iPSCs to self-renew and proliferate faster, and greatly reduce the passage time of iPSCs. In addition, iPSCs not only have improved proliferation efficiency, but also can maintain a good survival state, which directly promotes the significant increase in the total number of cells, and provides a more sufficient source of high-quality cells for subsequent applications such as cell therapy, drug screening, and disease model construction. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 This is a transmission electron micrograph of exosomes secreted by mouse embryonic fibroblasts prepared in Example;

[0015] Figure 2 A schematic diagram of the use of the cloning ring device used in the embodiment;

[0016] Figure 3 This is the gel electrophoresis image of dry gene identification by PCR after induced pluripotent stem cells were passaged 20 times in Example 2;

[0017] Figure 4 This is a cell karyotype analysis diagram of induced pluripotent stem cells after 20 passages in Example 2;

[0018] Figure 5 HE staining of teratoma after 20 passages of induced pluripotent stem cells in Example 2;

[0019] Figure 6The morphological diagram of iPSCs obtained by the culture methods of Examples 1 to 3 and Comparative Example 1;

[0020] Figure 7 The cell viability, cell number and passage time of the culture methods of Examples 1 to 3 and Comparative Example 1 are shown. DETAILED DESCRIPTION

[0021] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] It should be noted that:

[0023] In this document, unless otherwise specified, all implementation methods and preferred implementation methods mentioned in this document can be combined with each other to form a new technical solution.

[0024] In this document, unless otherwise specified, all technical features and preferred features mentioned in this document can be combined with each other to form a new technical solution.

[0025] Herein, unless otherwise specified, the components or preferred components involved can be combined with each other to form a new technical solution.

[0026] In this document, unless otherwise specified, the numerical range "a-b" is an abbreviation for any combination of real numbers between a and b, where a and b are both real numbers. For example, the numerical range "6-22" means that all real numbers between "6-22" are listed in this document, and "6-22" is just an abbreviation for these numerical combinations.

[0027]

[0046] Herein, a "range" disclosed in the form of a lower limit and an upper limit may be one or more lower limits, and one or more upper limits, respectively.

[0028] As used herein, unless otherwise stated, "optionally", "optional", "optional" or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.

[0029] Herein, the terms "comprise" or "comprising" are intended to include stated elements, integers or steps, but not to exclude any other elements, integers or steps.

[0030] In the present invention, unless otherwise specified, each reaction or operation step may be performed in sequence or not.

[0031] Unless otherwise specified, the professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content may also be applied to the present invention.

[0032] In a first aspect, a method for culturing induced pluripotent stem cells is provided. The culturing method comprises allowing the induced pluripotent stem cells to grow by attaching to a cell culture matrix, wherein the cell culture matrix contains exosomes. The cell culture matrix refers to the part in the space outside the cell that provides physical support for the cell, and optionally participates in biological processes such as cell attachment, migration, proliferation, differentiation and apoptosis.

[0033] In an optional embodiment, the concentration of exosomes in the cell culture matrix is ​​0.05-0.2 mg / mL based on the total protein concentration of the exosomes, for example, but not limited to 0.05, 0.1, 0.15 or 0.2 mg / mL. In an optional embodiment, the cell culture matrix is ​​coated on the surface of the culture container.

[0034] In an optional embodiment, the culture method comprises inoculating induced pluripotent stem cells on a surface coated with the cell culture matrix, and culturing the induced pluripotent stem cells after adding culture medium.

[0035] In an optional embodiment, the culture method includes inoculating induced pluripotent stem cells into the cell culture matrix, adding culture medium and then culturing the induced pluripotent stem cells. For example, the induced pluripotent stem cells are mixed with the cell culture matrix and then placed in a culture container for culture.

[0036] In an optional embodiment, the exosomes include exosomes derived from feeder cells, wherein the feeder cells refer to cells that can serve as a feeder layer during the in vitro culture of induced pluripotent stem cells, and exemplary feeder cells include but are not limited to embryonic fibroblasts or fibroblasts.

[0037] In an optional embodiment, the exosomes include exosomes secreted by embryonic fibroblasts, preferably exosomes secreted by mouse embryonic fibroblasts.

[0038] In an optional embodiment, the cell culture matrix includes a hydrogel, and the hydrogel can be selected from any hydrogel that is optional and acceptable in the art, and the present invention is not limited to this. The hydrogel includes, but is not limited to, a natural hydrogel or a synthetic hydrogel. Exemplary natural hydrogels include, but are not limited to, polysaccharides (e.g., hyaluronic acid, alginate, chitosan), proteins (e.g., collagen, fibrin), etc.; exemplary synthetic hydrogels include, but are not limited to, polyethylene glycol (PEG) hydrogels or polyacrylamide (PAAm) hydrogels, etc.

[0039] In an optional embodiment, the hydrogel includes a thermosensitive hydrogel. Thermosensitive hydrogels are hydrogels that can undergo sol-gel phase transitions as the ambient temperature changes. Exemplary thermosensitive hydrogels include, but are not limited to, poly-N-isopropylacrylamide (PNIPAM) hydrogels, chitosan thermosensitive hydrogels, LGA-PEG-PLGA hydrogels composed of poly-lactic-co-glycolic acid (PLGA) and polyethylene glycol (PEG), or block copolymer hydrogels of poly-N-isopropylacrylamide and polyethylene glycol.

[0040] In an optional embodiment, the thermosensitive hydrogel is in a liquid state at a temperature of 2-10° C. and in a gel state at a temperature of 37° C. In this embodiment, the phase transition temperature of the thermosensitive hydrogel can be any temperature between greater than 2° C. and less than 37° C.

[0041] In an optional embodiment, the culturing method includes mixing the thermosensitive hydrogel with exosomes when the thermosensitive hydrogel is in a liquid state, and then placing the thermosensitive hydrogel in an environment above the phase transition temperature to transform the thermosensitive hydrogel into a gel state.

[0042] In an optional embodiment, the culture method includes mixing the thermosensitive hydrogel with exosomes when the thermosensitive hydrogel is in a liquid state, and then adding the mixed system to a culture container, so that the thermosensitive hydrogel containing the exosomes is coated on the surface of the culture container.

[0043] In an optional embodiment, the culture method includes mixing the thermosensitive hydrogel with induced pluripotent stem cells when the thermosensitive hydrogel is in a liquid state, so that the induced pluripotent stem cells are seeded in the thermosensitive hydrogel.

[0044] In an optional embodiment, the culture method includes coating the thermosensitive hydrogel on the surface of a culture container when the thermosensitive hydrogel is in a liquid state, and inoculating the induced pluripotent stem cells on the surface of the thermosensitive hydrogel after the thermosensitive hydrogel is converted into a gel state.

[0045] Since the thermosensitive hydrogel can be converted into a liquid state within a temperature range, as long as the thermosensitive hydrogel in the culture environment maintains a temperature that can maintain a liquid state, the thermosensitive hydrogel can be converted into a liquid state, so that the cells can be separated from the culture container, thereby achieving the purpose of digesting the cells. Therefore, in an optional embodiment, a pre-cooled culture medium or buffer is used to digest the cells in the culture container, and the pre-cooling includes making the temperature of the culture medium or buffer consistent with the temperature at which the thermosensitive hydrogel maintains a liquid state. This digestion method can achieve cell digestion by simply changing the temperature, avoiding damage to cells by traditional digestion reagents.

[0046] In an optional embodiment, the culture method further comprises reprogramming somatic cells, wherein the somatic cells are inoculated according to any of the above-mentioned inoculation methods for induced pluripotent stem cells, so that the somatic cells adhere to the cell culture matrix and are reprogrammed to form induced pluripotent stem cell monoclonal colonies. Reprogramming can be performed by any method known in the art, including but not limited to using retrovirus, lentivirus, adenovirus, Sendai virus or plasmid to mediate cell reprogramming by transferring reprogramming factors into cells; or, using recombinant proteins including transcription factors to mediate cell reprogramming; or, introducing mRNA encoding reprogramming factors into cells.

[0047] The cells used to prepare induced pluripotent stem cells may be any partially differentiated or fully / terminally differentiated somatic cells acceptable in the art, and the present invention is not limited thereto.

[0048] In an optional embodiment, the induced pluripotent stem cells are obtained by reprogramming peripheral blood mononuclear cells (PBMC).

[0049] In an optional embodiment, after the monoclonal colony is formed, a cloning ring device is used to separate the monoclonal colony, and the separation of the monoclonal colony includes putting the ring body of the cloning ring on the monoclonal colony to be separated, adding a digestion reagent into the ring body, and separating the cells in the ring body into a new culture system after the cells detach from the culture container.

[0050] In an optional embodiment, when the cell culture matrix is ​​the above-mentioned temperature-sensitive hydrogel, the digestion reagent is the above-mentioned pre-cooled culture medium or buffer.

[0051] In an optional embodiment, a cloning ring device is further optimized, and the optimized cloning ring device includes a ring body and a flexible hollow container sealed and connected to the ring body, and the discharge port of the flexible hollow container is connected to the ring body. The connection includes being in a connected state all the time, or the discharge port of the flexible hollow container is connected to the ring body under specific conditions.

[0052] The ring body is used to cover the monoclonal colony therein, so that the monoclonal colony therein is separated from the culture environment.

[0053] The flexible hollow container is pre-loaded with a digestion reagent, or the digestion reagent is sucked into the container during use. After applying pressure to the flexible hollow container, the digestion reagent in the flexible hollow container is released into the ring body and contacts the monoclonal colony. Since negative pressure is formed in the cloning ring device after the digestion reagent in the flexible hollow container is squeezed out, after the pressure applied to the flexible hollow container is released, the digested cells are sucked into the flexible hollow container, so that the cells in the ring body are separated from the original culture medium.

[0054] The digestion reagent can be kept inside the flexible hollow container by sucking in the negative pressure formed by squeezing the flexible hollow container; a barrier can also be set at a position where the discharge port is connected to the ring body, so that the digestion reagent is pre-installed in the flexible hollow container. When pressure is applied to the flexible hollow container, the barrier opens and the digestion reagent therein enters the ring body. After the pressure is removed, the barrier closes, or the barrier is destroyed after the first pressure is applied.

[0055] The ring body can delimit the monoclonal colony, and its cross-sectional shape is not limited, and its cross-section can be, but is not limited to, a circular ring, a polygon, a rectangle or an irregular shape. In an optional embodiment, the ring body is a ring column.

[0056] In an optional embodiment, the longitudinal cross-section of the flexible hollow container is rectangular.

[0057] The optimized cloning ring device in the above embodiment helps to quickly pick up single clone cells.

[0058] The liquid culture medium, culture medium additives and culture conditions used in each culture stage in the culture method of induced pluripotent stem cells provided in the first aspect can be carried out according to methods well known in the art and recorded in various general and more specific textbooks, references, process manuals, product descriptions and standard documents, etc., and the present invention is not limited thereto.

[0059] In a second aspect, a kit for inducing pluripotent stem cell culture is also provided, the kit comprising a thermosensitive hydrogel, exosomes and a culture medium for inducing pluripotent stem cells.

[0060] In an optional embodiment, based on the total protein concentration of the exosomes, the working concentration of the exosomes is 0.05-0.2 mg / mL, for example, but not limited to 0.05, 0.1, 0.15 or 0.2 mg / mL.

[0061] In an alternative embodiment, the exosomes include exosomes derived from feeder cells.

[0062] In an optional embodiment, the exosomes include exosomes secreted by embryonic fibroblasts or fibroblasts, preferably exosomes secreted by mouse embryonic fibroblasts.

[0063] In an optional embodiment, the exosomes in the kit are premixed in the thermosensitive hydrogel.

[0064] In an optional embodiment, the kit includes a culture container, the surface of which is used to attach cells and is coated with a thermosensitive hydrogel containing exosomes.

[0065] In an optional embodiment, the kit further comprises the optimized cloning ring device described in the first aspect.

[0066] In an optional embodiment, a reprogramming reagent is also included, and the reprogramming reagent includes but is not limited to retrovirus, lentivirus, adenovirus, Sendai virus, plasmid, transcription factor recombinant protein or polynucleotide encoding reprogramming factor.

[0067] In a third aspect, there is provided a method for culturing induced pluripotent stem cells as described in the first aspect, or a kit for culturing induced pluripotent stem cells as described in the second aspect, and use thereof in the preparation of a cell medicine.

[0068] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0069] The exosomes used in the following examples are mouse embryonic fibroblast exosomes, which were prepared according to the following method:

[0070] (1) Culture of mouse embryonic fibroblasts

[0071] Mouse embryonic fibroblasts were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The cells were resuspended in DMEM + 10% FBS (Gbico, USA) medium and inoculated into culture bottles, and cultured overnight in a 37°C, 5% CO2 incubator. When the cells were 80% to 90% confluent, they were digested with 0.25% trypsin and subcultured at a ratio of 1:3.

[0072] (2) Extraction of exosomes from mouse embryonic fibroblasts

[0073] Mouse embryonic fibroblasts were cultured at 6000 / cm 2 The density was inoculated into a T175 culture flask and cultured in a 37°C, 5% CO2 incubator. When the mouse embryonic fibroblasts grew to about 90% confluence, the culture supernatant was collected and centrifuged at 4°C, 2000g for 10 minutes to obtain the supernatant. After sterile filtration with a 0.22μm filter, it was concentrated to 10mL using a 100kDa MW ultrafiltration tube. Centrifuge at 4°C, 100,000g for 1h, discard the supernatant, and add 0.5mL PBS to resuspend. Repeat this step. After sterile filtration with a 0.22μm filter membrane, the PBS was resuspended to obtain a suspension containing exosomes and identified. PBS was adjusted to different concentrations of 0.20-0.25mg / mL (total protein concentration) to obtain the mouse embryonic fibroblast exosome preparation, which was transferred into a cryopreservation tube and stored in a liquid nitrogen tank for later use. All the above operations were performed in a biological safety cabinet. The transmission electron microscopy image of the exosomes secreted by the prepared mouse embryonic fibroblasts is shown in the following figure Figure 1 shown.

[0074] The schematic diagram of the cloning ring device used in the following examples is as follows Figure 2As shown, Figure 2 1 is a ring body, and 2 is a flexible hollow container. The cloning ring device used in the following embodiments consists of a ring body 1 and a flexible hollow container 2 sealed and connected to the ring body 1. The discharge port of the flexible hollow container 2 is connected to the inside of the ring body 1. Pre-cooled culture medium or buffer is inhaled during use. After the culture fluid is poured out of the cell culture dish, the monoclonal colony is covered with the ring body 1, and then the pre-cooled culture medium or buffer is squeezed out from the flexible hollow container 2. When the thermosensitive hydrogel is in liquid state, it is sucked out with the flexible hollow container 2 and transferred to a new culture bottle or culture dish.

[0075] Example 1

[0076] A method for culturing iPSCs is provided, comprising the following steps:

[0077] 1. Preparation of thermosensitive hydrogels capable of carrying exosomes

[0078] (1) Preparation of thermosensitive hydrogel

[0079] The preparation method comprises the following steps:

[0080] Add induced pluripotent stem cell complete medium mTesR (Stemcell, USA) to freeze-dried thermosensitive gel (Cosmo Bio, Japan) in a culture bottle, place the culture bottle at 4°C for about 3 hours, the freeze-dried thermosensitive gel will slowly absorb the culture medium, shake the bottle gently, and after overnight at 4°C, the freeze-dried thermosensitive gel will completely dissolve to obtain a thermosensitive sol. The thermosensitive sol will automatically convert into a gel state at 37°C.

[0081] (2) Configuring thermosensitive glue loaded with exosomes

[0082] Take the thermosensitive sol obtained in step (1), add exosomes secreted by mouse embryonic fibroblasts into it in an ice bath at 0-4°C, mix the system with a pipette, adjust the final concentration of exosomes to 0.05 mg / mL, and obtain a thermosensitive hydrogel loaded with exosomes. The thermosensitive hydrogel containing exosomes gels at 37°C.

[0083] 2. Induction of PBMC into iPSCs

[0084] Thermosensitive gel loaded with exosomes is added to a 6-well plate at 2-10°C and placed in a 37°C, 5% CO2 incubator. After solidification, cells can be inoculated and cultured in the gel state of the thermosensitive gel.

[0085] Take 50 mL of fresh blood sample, extract it with ficoll separation solution, and inoculate PBMC cells into 6-well plates coated with thermosensitive hydrogel containing 0.05 mg / mL exosomes, and then use Sendai virus 2.0 system reprogramming kit (ThermoFisher, USA). Figure 2 As shown in the figure, pre-cooled iPSCs expansion medium was added to allow the clones to detach from the bottom of the dish and transfer to a culture dish coated with a thermosensitive hydrogel containing 0.05 mg / mL of exosomes for subculture.

[0086] 3. Culture of induced pluripotent stem cells

[0087] When the cell density reaches 80%, discard the culture supernatant, add 1mL of pre-cooled D-PBS to detach the cells from the bottom of the dish, transfer to a 15mL sterile centrifuge tube, and then add 10mL of D-PBS to mix gently. Centrifuge at 300g for 5min and discard the supernatant. Add 2mL of room temperature mTesR medium and mix gently, then add the cell suspension to the thermosensitive hydrogel coated with 0.05mg / mL exosomes at a ratio of 1:10. Culture in a 37℃, 5% CO2 incubator until the cell confluence rate reaches about 80% again, then it can be passaged.

[0088] From the 5th to the 10th generation of cells, the cell viability, number and time required for subculturing were recorded (starting from the time the well plate was taken out of the incubator to the time it was transferred to a 15 mL centrifuge tube, and the integer was taken).

[0089] Example 2

[0090] Provided is an iPSC culture method, which differs from Example 1 only in that the exosome content in the thermosensitive hydrogel is 0.1 mg / mL.

[0091] Example 3

[0092] Provided is an iPSC culture method, which differs from Example 1 only in that the exosome content in the thermosensitive hydrogel is 0.2 mg / mL.

[0093] Comparative Example 1

[0094] Provided is an iPSC culture method, which differs from Example 1 only in that a plate coated with VTN protein (Stemcell, USA) is used in steps 2 and 3, and 0.5 mM EDTA (Shownin, China) is used for digestion for 8 minutes and passage.

[0095] Effect Example 1

[0096] Stemness gene detection of induced pluripotent stem cells

[0097] The iPSCs precipitate cultured in Example 2 (thermosensitive hydrogel containing 0.1 mg / mL exosomes) at the 20th generation was taken, RNA was extracted, and reverse transcribed into cDNA. The operation was performed according to the operating instructions of the real-time quantitative PCR kit (ThermoFisher, USA), and the expression levels of stemness genes SOX2, OCT4, and Nanog were mainly detected. The reaction system was configured as follows:

[0098] Table 1. PCR reaction system

[0099] 2×Mix(SYBR Green) 10μL 50×ROX Reference Dye 0.4μL Forward primer (10 μM) 0.4μL Reverse primer (10 μM) 0.4μL cDNA template 1μL <![CDATA[RNase-Free ddH2O]]> 7.8μL

[0100] Except for cDNA, mix the above system components evenly, add them to 8-tube strips, 19μL per well, and finally add 1μL cDNA sample. Cover the tube cap, centrifuge and put it into the ABI7500 instrument. Set the reaction program: pre-denaturation 95℃, 2min; 95℃, 30s; 65℃, 30s; 72℃, 30s; 35 cycles in total. Take 5μL of PCR product and 1μL of 6× DNA loading buffer and mix them thoroughly for agarose gel electrophoresis. After 120V constant voltage electrophoresis for 20 minutes, observe and take pictures in the gel imaging system. PBMCs that were not infected with Sendai virus were used as the control group to compare the expression of stemness genes in induced pluripotent stem cells. The experimental results are as follows. Figure 3 As shown, the reprogrammed cells expressed the key transcription factors NANOG, OCT4, and SOX2 genes of iPSCs, indicating that PBMCs were successfully reprogrammed into iPSCs.

[0101] Effect Example 2

[0102] Karyotype analysis of induced pluripotent stem cells

[0103] iPSCs cultured in Example 2 (thermosensitive hydrogel containing 0.1 mg / mL exosomes) at the 20th generation were inoculated into a T75 culture flask, and when the cells grew to a confluence of about 80%, they were sent to Hangzhou Aidikang Medical Testing Center for karyotype detection. The results showed that ( Figure 4 ), 24 cells were randomly analyzed under a microscope, and all the division phases had 46 chromosomes, and the chromosome morphology was normal. The results showed that after 20 generations of passage by the passage method of Example 2, the karyotype of the induced pluripotent stem cell line could still maintain normal, indicating that this passage method is suitable for the passage of induced pluripotent stem cells.

[0104] Effect Example 3

[0105] In vivo differentiation of teratomas from induced pluripotent stem cells

[0106] Take the iPSCs cultured in Example 2 (thermosensitive hydrogel containing 0.1 mg / mL exosomes) at the 20th generation and collect the cells. The cells in one well of a six-well plate were taken as a sample, resuspended with 200 μL mTesR complete medium, 2 μL ROCK inhibitor and 200 μL of matrix gel, and placed on ice. Male SCID mice aged 6 to 8 weeks were randomly selected, and the cell suspension was injected subcutaneously into the back and legs of each mouse, with 100 μL injected into each site. The injected mice were raised separately in separate cages, and the growth of teratomas was observed every week. When the teratoma grows (the diameter of the tumor does not exceed 1 cm), it is quickly killed by cervical dissection, and the tumor is dissected out and fixed overnight in 4% paraformaldehyde solution. The samples were sent to a third-party testing agency for HE staining to observe whether the three-germ layer structure can be formed. The HE staining results are as follows Figure 5 As shown, it can be seen that the tumor has formed a three-germ layer structure.

[0107] Effect Example 4

[0108] The morphology of iPSCs obtained by the culture methods of Examples 1 to 3 and Comparative Example 1 is shown in the figure below. Figure 6 As shown, it can be seen that the iPSCs obtained by the culture methods of Examples 1 to 3 are no different from those of Comparative Example 1. Comparison of cell viability, cell number and passage time of Examples 1 to 3 and Comparative Example 1 shows that Figure 7 As shown, it can be seen that the cell viability of iPSCs cultured by the methods of Examples 1 to 3 is comparable to that of the existing method of Comparative Example 1, but the cell number of iPSCs is significantly better than that of Comparative Example 1, and the passage time is shorter, indicating that the culture methods of Examples 1 to 3 are better than the method of Comparative Example 1.

[0109] The above experimental results show that after 20 generations of passage using the method of the embodiment, the number of cells is large, the karyotype of human induced pluripotent stem cells has not changed, and they express pluripotency-related genes and have the ability to differentiate into different germ layer tissues.

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for culturing induced pluripotent stem cells, It is characterized in that The method comprises allowing induced pluripotent stem cells to adhere to and grow on a cell culture matrix, wherein the cell culture matrix contains exosomes.

2. The culture method according to claim 1, It is characterized in that The concentration of exosomes in the cell culture matrix is ​​0.05-0.2 mg / mL based on the total protein concentration of the exosomes; Optionally, the exosomes include exosomes derived from feeder cells; Optionally, the exosomes include exosomes secreted by embryonic fibroblasts or fibroblasts.

3. The culture method according to claim 1, It is characterized in that The cell culture matrix comprises a hydrogel.

4. The culture method according to claim 3, It is characterized in that The hydrogel includes a thermosensitive hydrogel; Optionally, the thermosensitive hydrogel is in liquid state at a temperature of 2 to 10°C and in gel state at a temperature of 37°C; Optionally, cells are digested using a pre-cooled culture medium or buffer, wherein the pre-cooling includes making the temperature of the culture medium or buffer consistent with the temperature at which the thermosensitive hydrogel maintains a liquid state.

5. The culture method according to claim 1, It is characterized in that The culture method further comprises reprogramming somatic cells, wherein the somatic cells adhere to the cell culture matrix and are reprogrammed to form monoclonal colonies of induced pluripotent stem cells.

6. The culture method according to any one of claims 1 to 5, It is characterized in that The method comprises using a cloning ring device to separate monoclonal colonies, wherein the separation of monoclonal colonies comprises putting a ring body of the cloning ring on the monoclonal colony to be separated, adding a digestion reagent into the ring body, and separating the cells in the ring body into a new culture system after the cells are separated from the culture container; Optionally, the cell culture matrix is ​​a temperature-sensitive hydrogel, and the digestion reagent is the pre-cooled culture medium or buffer as described in claim 4.

7. The culture method according to claim 6, It is characterized in that The cloning ring device comprises a ring body and a flexible hollow container sealedly connected to the ring body for storing a digestion reagent, wherein a discharge port of the flexible hollow container is communicated with the inside of the ring body.

8. A kit for inducing pluripotent stem cell culture, It is characterized in that Comprising a thermosensitive hydrogel, exosomes, and a culture medium for inducing pluripotent stem cells.

9. The kit according to claim 8, It is characterized in that Based on the total protein concentration of exosomes, the working concentration of exosomes is 0.05-0.2 mg / mL; Optionally, the exosomes include exosomes derived from feeder cells; Optionally, the exosomes include exosomes secreted by embryonic fibroblasts or fibroblasts; Optionally, the kit includes a culture container, the surface of the culture container for attaching cells is coated with a thermosensitive hydrogel containing exosomes; Optionally, the kit further comprises the cloning ring device as described in claim 7; Optionally, the kit further comprises a reprogramming reagent.

10. Use of the method for culturing induced pluripotent stem cells according to any one of claims 1 to 7, or the kit for culturing induced pluripotent stem cells according to claim 8 or 9 in the preparation of a cell medicine.

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