A simple and rapid method for preparing patterned cell films

Abstract

The present invention belongs to the technical field of cell regenerative medicine, and discloses a method for simply and quickly preparing a patterned cell film. Specifically, a method for preparing a patterned cell film is disclosed, which comprises the following steps: placing sealing films with different pattern properties close to the bottom of a container, inoculating cells, culturing, removing the sealing films, and obtaining patterned cell films. The present invention realizes patterning of cell films by controlling the shape and size of the sealing films. Compared with the traditional method, there is no need for special processing of custom culture dishes or cell culture auxiliary molds. The sealing films used are common experimental supplies and are inexpensive, and patterns can be set according to actual needs.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell regenerative medicine, and in particular relates to a method for simply and quickly preparing a patterned cell film. Background Art

[0002] Cell membrane technology is a scaffold-free tissue engineering technology with broad application prospects in the fields of tissue engineering and regenerative medicine. Cell membranes are composed of cells and extracellular matrix, with high cell density. They can completely retain the binding proteins between cells and the adhesive proteins between cells and the base, effectively maintaining cell-to-cell interactions. In addition, a small amount of extracellular matrix can play a role similar to glue, which is conducive to bonding and lamination, and facilitates subsequent tissue repair and the construction of three-dimensional models.

[0003] At present, temperature-sensitive culture dishes are commonly used to prepare cell films. The surface of the temperature-sensitive culture dish is coated with poly(N-isopropylacrylamide) [poly(N-isopropylacrylamide), PIPAAm]. When the temperature is lower than 32°C, PIPAAm changes from hydrophobic to hydrophilic, causing the cells on the culture dish to fall off by themselves and form a complete cell film. The shape of the cell film peeled off from the temperature-sensitive culture dish is consistent with the shape of the culture dish, usually circular, but in actual applications, there will be different shape requirements for different scenarios. The traditional method is to customize a temperature-sensitive culture dish of a specific shape or customize a mold of a specific shape, fix the mold to the bottom of the culture dish, and then inoculate cells into the mold. There are also literature reports on the use of gratings on temperature-sensitive culture dishes, through light-induced chemical grafting of polyacrylamide, and the part without grafted polyacrylamide can be attached to cells, thereby realizing the patterned regulation of cell films. It is found that the existing methods for making patterned cell films require special customized molds or more complex chemical methods, which are time-consuming and labor-intensive, complicated to operate and costly. Summary of the invention

[0004] The first aspect of the present invention aims to provide a method for preparing a patterned cell film.

[0005] The purpose of the second aspect of the present invention is to provide an application of the patterned cell membrane prepared by the preparation method of the first aspect of the present invention or the preparation method of the patterned cell membrane of the first aspect of the present invention in wound repair.

[0006] The purpose of the third aspect of the present invention is to provide a method for preparing the patterned cell film of the first aspect of the present invention or to use the patterned cell membrane obtained by the method for preparing the patterned cell film of the first aspect of the present invention in constructing bionic tissue.

[0007] The purpose of the fourth aspect of the present invention is to provide a method for preparing the patterned cell film of the first aspect of the present invention or to use the patterned cell membrane obtained by the method for preparing the patterned cell film of the first aspect of the present invention in the safety evaluation and toxicity detection of biomaterials or drugs.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention provides a method for preparing a patterned cell film, comprising the following steps: attaching sealing films with different patterns to the bottom of a container, inoculating cells, culturing, and removing the sealing films to obtain a patterned cell film.

[0010] Preferably, the sealing film is sterilized before being attached to the container.

[0011] Preferably, the sterilization treatment includes alcohol solution immersion and ultraviolet irradiation.

[0012] Preferably, the container comprises a cell culture dish.

[0013] Preferably, the cell culture dish comprises a temperature-sensitive culture dish and a cell culture dish.

[0014] Preferably, the cell inoculation number is 80,000 to 150,000 / cm 2 ; preferably 100,000 to 150,000 / cm 2 More preferably 150,000 / cm 2 .

[0015] Preferably, the cells are obtained by cell recovery or conventional culture passaging.

[0016] Preferably, the cell is derived from human.

[0017] Preferably, the cells include at least one of fibroblasts and bone marrow mesenchymal stem cells.

[0018] Preferably, the culture medium comprises serum, double antibodies and basal culture medium.

[0019] Preferably, the basal culture medium comprises at least one of DMEM and alpha-MEM.

[0020] Preferably, the culture conditions are 30-37° C. and 3%-7% CO 2 concentration for 3-10 days.

[0021] More preferably, the culture conditions are 35-37° C. and 3%-5% CO 2 concentration for 5-8 days.

[0022] More preferably, the culture conditions are 35-37° C. and 3%-5% CO 2 concentration for 5-8 days.

[0023] Preferably, during the culture process, the medium is replaced every 2 to 3 days.

[0024] Preferably, the preparation method further comprises peeling.

[0025] Preferably, when a temperature-sensitive culture dish is used, the stripping comprises the following steps: placing the container with the sealing film removed at 10-32° C. for 30-45 min; when an ordinary cell culture dish is used, gently blowing the edge of the container with a pipette.

[0026] Further preferably, when a temperature-sensitive culture dish is used, the stripping comprises the following steps: placing the container with the sealing film removed at 15-25° C. for 30-45 minutes.

[0027] The second aspect of the present invention provides the preparation method of the first aspect of the present invention or the use of the patterned cell membrane prepared by the preparation method of the first aspect of the present invention in preparing a wound repair product.

[0028] The third aspect of the present invention provides a method for preparing the patterned cell membrane according to the first aspect of the present invention or use of the patterned cell membrane prepared by the preparation method according to the first aspect of the present invention in constructing bionic tissue.

[0029] Preferably, the bionic tissue includes bionic vascular tissue and bionic bone tissue.

[0030] The fourth aspect of the present invention provides a method for preparing the patterned cell film according to the first aspect of the present invention or the use of the patterned cell membrane prepared by the preparation method of the first aspect of the present invention in the safety evaluation and toxicity detection of biomaterials or drugs.

[0031] The beneficial effects of the present invention are:

[0032] The present invention realizes patterned processing of cell membranes by controlling the shape and size of the sealing film. Compared with the traditional method, there is no need for special processing and customization of culture dishes or cell culture auxiliary molds. The sealing film used is a common experimental product and is low in price, and the pattern can be set according to actual needs.

[0033] The preparation method provided by the present invention is generally applicable to any cell culture dish and any adherent cells. The obtained patterned cell film has a wide range of application scenarios and is expected to be used to construct small-caliber artificial blood vessels, in vitro bionic tissues or organs, and personalized "band-aids" for wound repair, so that cell film technology can be better applied to the fields of tissue engineering and regenerative medicine.

[0034] The cell film obtained by the preparation method provided by the present invention is directly applied to the wound site, so that most of the cells are retained in the lesion site, and are used for wound repair and promote wound healing through the effect of cell paracrine. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 are patterned cell membranes; among them, A is a long strip cell membrane, left: before peeling, right: after peeling; B is a triangular cell membrane, left: before peeling, right: after peeling; C is a square cell membrane, left: before peeling, right: after peeling; D is a four-leaf clover-shaped cell membrane, left: before peeling, right: after peeling.

[0036] Figure 2 The long strip cell membrane of Example 1 is rolled into a tubular tissue.

[0037] Figure 3 This is the "coin-shaped" cell film stack of Example 2.

[0038] Figure 4 The figure shows the results of using the square cell membrane and the triangular cell membrane of Example 3 for wound repair in rats. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below through specific examples.

[0040] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0041] Unless otherwise specified, the materials and reagents used in this example were obtained from commercial sources.

[0042] Example 1 Long strip cell membrane / NHDF / temperature sensitive culture dish

[0043] A method for preparing a long strip of NHDF cell film comprises the following steps:

[0044] (1) According to the inner diameter of the bottom of a 35mm temperature-sensitive culture dish (purchased from CellSeed), draw a circle with a diameter of about 34mm on a sealing film (purchased from SEP), cut the circle with scissors, and then draw a rectangle with a diagonal length of 30mm in the circular sealing film. Cut off the rectangle in the middle of the circle to form a circular sealing film with a hollow center. Soak the hollow circular sealing film in 75% alcohol and expose it to ultraviolet light overnight. The next day, remove the sealing film from the alcohol and expose it to ultraviolet light until it is air-dried. Stick the hollow circular sealing film to the bottom of the temperature-sensitive culture dish, and press it gently with the head of the tweezers to make it fit completely with the bottom of the culture dish without leaving any gaps.

[0045] (2) Normal human skin fibroblasts (NHDF) were revived and passaged. The 7th generation NHDF was inoculated on a temperature-sensitive culture dish with a sealing film on the bottom at a density of 1 million / dish. 2 mL of culture medium (low-glucose DMEM containing 10 v / v% fetal bovine serum and 1 v / v% double-antibody (penicillin-streptomycin) (purchased from Corning)) was added and cultured in a 37°C, 5% CO2 incubator for 6 days. The medium was replaced every 2 days.

[0046] (3) After 6 days of culture, the temperature-sensitive culture dish was taken out of the incubator and the sealing film at the bottom of the culture dish was removed with tweezers. The temperature-sensitive culture dish was placed in a 20°C low-temperature incubator for 45 minutes to allow the NHDF to completely detach from the bottom of the culture dish, thus obtaining a long strip of NHDF cell film.

[0047] Use a plastic sheet to transfer the long strip of NHDF cell film to the round plastic tube so that it wraps around the outer periphery of the plastic tube. Figure 2 As shown, a tubular tissue is formed. This structure is expected to be used in the future to construct tubular bionic tissues such as artificial blood vessels to simulate artificial blood vessels in vitro.

[0048] Example 2 “Coin-shaped” cell membrane / hBMSC / temperature-sensitive culture dish

[0049] A method for preparing a copper coin-shaped hBMSC cell film comprises the following steps:

[0050] (1) According to the inner diameter of the bottom of a 35mm temperature-sensitive culture dish (purchased from CellSeed), draw a circle with a diameter of about 12mm on the sealing film (purchased from SEP), and cut the circle with scissors to form a circular sealing film. Soak the circular sealing film in 75% alcohol and irradiate it with ultraviolet light overnight. The next day, remove the sealing film from the alcohol and irradiate it with ultraviolet light until it is air-dried. Stick the circular sealing film to the center of the bottom of the temperature-sensitive culture dish, and press it gently with the head of the tweezers to make it fit completely with the bottom of the culture dish without leaving any gaps.

[0051] (2) hBMSC cells were revived and passaged. The sixth-generation cells were seeded on a temperature-sensitive culture dish with a sealing film on the bottom at a seeding density of 1 million / dish. 2 mL of culture medium (alpha-MEM (purchased from Corning) containing 10 v / v% fetal bovine serum and 1 v / v% double-antibody (penicillin-streptomycin)) was added and cultured in a 37°C, 5% CO2 incubator for 6 days. The medium was replaced every 2 days.

[0052] (3) After 5 days of culture, take the culture dish out of the incubator and remove the sealing film at the bottom of the dish with tweezers. Place the temperature-sensitive dish in a 20°C low-temperature incubator and incubate for 30 minutes to allow the cells to completely detach from the bottom of the dish, thus obtaining a "coin-shaped" cell film with a defect in the middle.

[0053] The copper coin-shaped hBMSC cell membranes were stacked for a total of 3 layers, as shown in Figure 3 As shown, a three-dimensional tissue with a defect in the middle can be formed. This tissue is expected to be used to construct a bone defect model, simulate human periosteal tissue with a defect in vitro, and then evaluate bone repair materials or drugs in vitro.

[0054] Example 3 Arbitrary shape of cell membrane / NHDF / ordinary cell culture dish

[0055] A method for preparing a square NHDF cell film comprises the following steps:

[0056] (1) According to the inner diameter of the bottom of a 35mm ordinary cell culture dish (purchased from Corning), draw a circle with a diameter of about 34mm on the sealing film (purchased from SEP), cut the circle with scissors, and then draw a square with a diagonal length of 30-32mm in the circular sealing film (it can also be an equilateral triangle with a side length of 29mm or a four-leaf clover with a long axis of symmetry of 32mm). Cut off the shape in the middle of the circle to form a circular sealing film with a hollow center. Soak the hollow circular sealing film in 75% alcohol and irradiate it with ultraviolet light overnight. The next day, remove the sealing film from the alcohol and irradiate it with ultraviolet light until it is air-dried. Stick the hollow circular sealing film to the bottom of the ordinary cell culture dish and press it gently with the head of the tweezers to make it fit completely with the bottom of the culture dish without leaving any gaps.

[0057] (2) NHDF was revived and passaged. The 7th generation cells were inoculated on a common cell culture dish with a sealing film on the bottom at a density of 1 million / dish. 2 mL of culture medium (special culture medium for NHDF cell films: DMEM high-glucose medium containing 10 v / v% fetal bovine serum, 1 v / v% penicillin-streptomycin, 1 v / v% ITS cell culture supplement, 0.04 μg / mL dexamethasone, 50 μg / mL vitamin C, 25 mg / mL Ficoll 400) was added and cultured in a 37°C, 5% CO2 incubator for 6 days. The medium was replaced every 2 days.

[0058] (3) After 6 days of culture, the NHDF cells were taken out of the incubator and the sealing film at the bottom of the culture dish was removed with tweezers. About 1 mL of culture medium was taken with a pipette and the bottom of the culture dish was gently blown to completely separate the cells from the bottom of the culture dish, thus obtaining a square (triangular or four-leaf clover) NHDF cell film.

[0059] Effect Example

[0060] According to the sizes and shapes of the square and triangular NHDF cell films obtained in Example 3, skin incisions with corresponding shapes were created on the back of the rat, with the side length of the square wound being 1.2 cm and the side length of the triangular wound being 1.2 cm.

[0061] The specific operation is as follows: the rats were anesthetized, and surgical forceps were used to cut a square wound with a side length of 1.2 cm and a triangular wound with a side length of 1.2 cm on the back of the rats. The rats were further divided into two groups, a treatment group and a control group. The square NHDF cell film and the triangular NHDF cell film obtained in Example 3 were pasted on the wounds of the rats in the treatment group; the control group did not receive any treatment. The treated rats were routinely raised, and the repair of the back wounds of the rats was observed on the 1st, 5th, 8th and 13th days, and photographed and recorded.

[0062] like Figure 4 As shown in the figure, on the 5th day of repair, there was no significant difference between the treatment group and the control group; but on the 8th day, it can be seen that the wound size of the treatment group was significantly smaller than that of the control group; on the 13th day, the wound of the treatment group was basically healed, while obvious scabs could be seen in the control group. Therefore, in practical applications, the morphology of the cell membrane can be adjusted according to the size and shape of the wound, thereby achieving personalized wound repair.

[0063] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for preparing a patterned cell film, comprising the following steps: The sealing films with different patterns are attached to the bottom of the container, cells are inoculated, cultured, and the sealing films are removed to obtain patterned cell films; The container comprises a cell culture dish; The culture dishes include temperature-sensitive culture dishes and common cell culture dishes; The culture conditions are 30-37° C. and 3%-7% CO2 concentration for 3-10 days; The sealing film is sterilized before being placed against the container; The preparation method further comprises peeling; When a temperature-sensitive culture dish is used, the stripping comprises the following steps: The container with the sealing film removed is placed at 10-32°C for 30-45 minutes. When a common cell culture dish is used, the stripping comprises the following steps: gently blowing the edge of the container with a pipette; The cells are human skin fibroblasts or human bone marrow mesenchymal stem cells.

2. The preparation method according to claim 1, characterized in that: The inoculation number of the cells is 80,000 to 150,000 / cm 2 .

3. Use of the preparation method according to claim 1 or 2 in preparing wound repair products.

4. Use of the preparation method according to claim 1 or 2 in constructing bionic tissue.

Images