WO2024087828A1 - Pet film for cell culture in cell and gene therapy, and use thereof - Google Patents

Pet film for cell culture in cell and gene therapy, and use thereof Download PDF

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WO2024087828A1
WO2024087828A1 PCT/CN2023/113930 CN2023113930W WO2024087828A1 WO 2024087828 A1 WO2024087828 A1 WO 2024087828A1 CN 2023113930 W CN2023113930 W CN 2023113930W WO 2024087828 A1 WO2024087828 A1 WO 2024087828A1
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pet film
cells
cell
cell culture
culture
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PCT/CN2023/113930
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French (fr)
Chinese (zh)
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谭虎
赵丹瑞
王江凯
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同腾新创(苏州)科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
    • C08J5/18—Manufacture of films or sheets
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2367/00—Characterised by the use of polyesters obtained by reactions forming a carboxylic ester link in the main chain; Derivatives of such polymers
    • C08J2367/02—Polyesters derived from dicarboxylic acids and dihydroxy compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers

Definitions

  • the invention relates to the technical field of cell culture, and in particular to a PET film used for cell culture in cell and gene therapy and an application thereof.
  • CGT Cell therapy and gene therapy
  • Cell culture technology is widely used in biological research, disease diagnosis, tissue engineering, drug screening and other technical fields. This technology is also the technical core for realizing large-scale production of CGT.
  • the PET (Polyethyleneterephthalate) membrane used in the 3D cell culture system is a new type of cell culture material that provides a three-dimensional environment for the growth and reproduction of adherent cells. Traditional 2D cell culture vessels are quite different from the actual growth environment of cells in the body, which causes cell growth characteristics and functions to change to a certain extent. PET membrane is an experimental material that is closer to the physiological state in the body. This 3D cell culture system better simulates the microenvironment of cell growth in the body and is easy to scale up.
  • 3D cell culture is mainly a scaffold-free culture system, that is, microspheres or microcarrier materials are formed by self-assembly of cells under the action of gravity through hanging drops.
  • these methods have high experimental operation complexity, expensive equipment and consumables, and personnel need additional technical training.
  • the amount of cell inoculation required is large. Therefore, in the field of cell culture, there is a need for a simple, efficient, cost-effective experimental material suitable for large-scale culture of adherent cells.
  • the present invention aims to provide a PET film for cell culture in cell and gene therapy, and the specific scheme is as follows:
  • PET film for cell culture in cell and gene therapy, wherein the PET film is a long-axis rolled film, which is spirally rolled to form a cylindrical film column as a whole, wherein the structure of the PET film is a regular crisscrossed membrane network support structure;
  • the hardening value of the film surface of the PET film after hardening treatment is between 3H and 4H;
  • the fibers inside the PET film are in a grid shape, and the fiber diameter is between 20-30 ⁇ m;
  • the tensile strength of the PET film is in the range of 3-6.5MPa
  • the PET film is hydrophilic
  • the PET film is transparent and colorless as a whole, and its light transmittance is greater than 90%.
  • the cylindrical multilayer membrane structure formed by spiral curling of the densely structured PET membrane has a good interception effect on cells.
  • the thick cell suspension can pass through the pores of the membrane, and the cells are evenly fixed in the three-dimensional 3D culture system to achieve uniform distribution.
  • the fibers of the PET membrane have a grid-like porous structure with low mass transfer resistance, which is conducive to the shuttling flow of liquid between the pores.
  • the culture medium flow can smoothly pass through the membrane network and fully contact the cells without dead corners, so that the cells can fully contact the culture medium in all directions and absorb a balanced amount of nutrients during the expansion stage, so the cell growth state is more uniform and the cell survival rate is higher.
  • the PET membrane can be used to culture adherent cells including mammalian cells, somatic cells, stem cells, progenitor cells, mature cells, fibroblasts, bone tissue, myocardium and smooth muscle, liver, lung, kidney, breast skin, glial cells, endocrine cells, melanocytes and various tumor cells.
  • mature cells may include neural cells, cardiomyocytes, retinal cells, and hepatocytes;
  • Stem cells may include mesenchymal stem cells, iPS cells, ES cells, Muse cells, embryonic cancer cells, and embryonic germ stem cells.
  • the working process of the PET film for cell attachment and culture is as follows:
  • the PET membrane that has passed various parameter tests is placed in a well plate, and after adding an appropriate amount of culture medium, cells are inoculated.
  • the carrier is transferred to a new well plate every 3 hours, and the unattached cells are collected and counted to calculate the attachment rate until the attachment rate is close to 80%;
  • the PET film is subjected to alkali neutralization treatment, sterilization treatment, and mycoplasma detection and bacterial endotoxin detection;
  • the specific alkaline neutralization treatment is as follows: the PET film is completely immersed in a 0.5M NaOH solution for 2 hours, and then rinsed twice with ultrapure water. The first rinse is 2 hours, and then the ultrapure water is replaced and rinsed for a second time for 4 hours.
  • the sterilization treatment is as follows: the PET film after alkali neutralization treatment is placed in a high-pressure steam sterilizer at a pressure of 100 kPa and a temperature of 121°C, sterilized for 45 minutes and then dried;
  • the standard for mycoplasma testing is the absence of mycoplasma, and the standard for bacterial endotoxin testing is the endotoxin content of ⁇ 0.25EU/piece.
  • the corresponding container carrier can be a cell culture dish, a square bottle or a rotating bottle for cell culture, a cell factory, or a cell bioreactor.
  • the PET film when used for cell culture, the PET film can be cut into a plurality of small long sampling strips, and the long sampling strips are placed at different positions of the inner layer, the middle layer and the outer layer of the membrane column.
  • the PET film used for cell culture in cell and gene therapy is used as a carrier in cell culture.
  • the present invention has the following beneficial effects:
  • Existing adherent cell culture mainly includes two forms: 2D culture and 3D culture.
  • the 2D culture method is a planar culture method, and the area of its culture surface limits the expansion multiples of adherent cells, resulting in a low yield and failing to meet the market's demand for large-scale production.
  • the 3D culture system constructed by the multi-layer dense membrane network structure of the PET film of the present invention provides cells with a geometric multiple three-dimensional growth space, effectively avoiding the problem of contact inhibition during the culture process and reducing the expansion rate.
  • the cell expansion amount is much higher than that of the ordinary culture mode, and is suitable for large-scale production and process amplification.
  • the present invention solves the problem in the prior art that adherent cells cannot be cultured on a large scale in 3D.
  • the 3D cell culture system constructed by the scaffold structure of the PET membrane well mimics the microenvironment of cell growth in vivo, which has a more scientific reference significance for studying the physiological state and activity of cells.
  • Figure 1 is a schematic diagram of the molecular structure of PET.
  • FIG. 2 is a schematic diagram of the structure of a PET membrane suitable for large-scale cell culture.
  • FIG. 3 is a schematic diagram of the PET membrane scaffold structure and its effect on cell attachment.
  • FIG4 is a schematic diagram of the microstructure of the PET film.
  • Figure 5 is a direct view of the porosity of the PET film under an electron microscope.
  • FIG. 6 is a tensile curve diagram of the PET film.
  • FIG. 7 is a schematic diagram of a material hydrophilicity and hydrophobicity test.
  • FIG. 8 is a schematic diagram showing the adhesion of HEK293T cells to the PET membrane.
  • PET stands for polyethylene terephthalate, which is produced by the condensation polymerization of terephthalic acid and ethylene glycol. It has good electrical properties, creep resistance, fatigue resistance, friction resistance, and material stability. The specific description is as follows:
  • PET has ester bonds, and its mechanical properties are little affected by high and low temperature environments, and the operating temperature is -100 to 120°C. It can withstand high temperature and high pressure sterilization, and the hydrophilicity of the material has been slightly improved after high pressure steam sterilization, because the trace amount of ester bonds are broken at high temperature to produce hydrophilic carboxyl and hydroxyl groups.
  • the gas and water vapor permeability is low, and it has excellent gas, water, oil and odor barrier properties.
  • Non-toxic, odorless, hygienic and safe suitable for medical packaging and equipment.
  • PET material is the lowest price among engineering plastics and has a high cost-effectiveness.
  • the present invention proposes a PET film for cell culture in cell and gene therapy.
  • the PET film is specifically an experimental material for 3D culture of adherent cells.
  • the material is PET (polyethylene terephthalate) film, the chemical formula is (C10H8O4)n, and the molecular structure diagram is shown in Figure 1.
  • the PET film of the present invention is a long-axis rolled film in terms of overall appearance and structure, which is spirally rolled to form a cylindrical film column as a whole (as shown in FIG. 2 ). When used, it can be flexibly adapted and cut according to the specifications of the cell culture container or carrier.
  • a scaffold is a commonly used culture matrix material that can provide physical support, and cells can enter the scaffold to grow and function (as shown in FIG3). Therefore, the structure of the PET film of the present invention is a regular crisscrossed membrane network scaffold structure from the internal structure, and the membrane network scaffold structure is used to intercept cells to achieve 3D culture of adherent cells. Therefore, the membrane network scaffold structure inside the PET film has biological safety, and the stability and mechanical properties are relatively good.
  • the surface structure, porosity and mechanical properties of the scaffold will also affect the migration behavior and growth state of cells.
  • Scaffolds can also be functionalized to give them different properties to further explore the 3D behavior of cells.
  • the present invention conducts a series of experimental tests on membrane structures of various PET materials, determines appropriate parameters, and screens out the PET membrane material that is most suitable for large-scale cell adherent culture.
  • This PET membrane is the content to be protected by the present invention.
  • the PET film needs to have certain gaps for the distribution of cells and culture medium.
  • the PET film material is fixed on the sample stage for gold spraying, and then observed with a scanning electron microscope.
  • the results are shown in Figure 4. From the microstructure of the PET film, it can be seen that the fibers of the PET film are evenly distributed in the field of view in a grid shape, forming a multi-void structure with low mass transfer resistance, which is conducive to the flow of culture medium. There are traces of melting at the intersection of the fibers, which are produced by melt-blowing method. There are few traces of hot melting. There is a membrane structure between the fibers, so the connection between the fibers is achieved by adhesives or cross-linking agents.
  • the fiber diameter and porosity of the PET film are key indicators that affect the probability of contact between cells and the culture surface.
  • a fiber diameter close to that of adherent cells (20 ⁇ m-50 ⁇ m) is conducive to cell attachment and spreading.
  • the fiber diameter of the PET film of the present invention is between 20-30 ⁇ m, which is conducive to cell attachment and implantation.
  • the present invention conducts a fiber diameter test experiment, and after screening multiple PET materials with different fiber diameters, selects a PET film material with a suitable fiber diameter. The specific method is as follows:
  • the electron microscope photos of different PET films are imported into ImageJ software. It should be noted that this software is the existing software that comes with the fiber diameter test experiment and does not involve improvement.
  • the area of the selected area in the entire photo is calculated to obtain the porosity of the material.
  • the processing results are shown in the three small pictures a, b, and c from left to right in Figure 5.
  • the porosities of the three PET film materials selected by calculation are 83%, 84%, and 76%, respectively.
  • the PET film with a porosity of 84% has strong cell adhesion and the highest cell adhesion rate.
  • the mechanical properties of the PET film are also one of the influencing indicators.
  • the present invention conducts a tensile strength test on the PET film, and the specific method is as follows:
  • PET film is cut into strip samples, the cross-sectional area is measured by thickness and width, and then the upper and lower ends of the sample are fixed on a tensile tester to measure the tensile curve of the sample.
  • a tensile tester to measure the tensile curve of the sample.
  • the tensile strength of the PET film is between 3-6.5MPa.
  • the PET film at this time has good mechanical properties, and folding resistance, creep resistance, fatigue resistance, and dimensional stability are all very good, which is most suitable for large-scale cell culture.
  • the wettability of the surface of solid materials is an important property of the surface of solid materials.
  • the surface wettability depends on the chemical composition and microscopic geometric structure of the material surface.
  • the droplet When a droplet falls on the horizontal surface of a solid material, the droplet may spread on the surface of the material or form a droplet at a certain angle and stop on the surface.
  • the contact angle At the junction of solid, liquid and gas, the angle from the solid-liquid interface through the liquid to the gas-liquid interface is called the contact angle ( ⁇ ).
  • the liquid When the contact angle is less than 90°, the liquid is said to be wettable to the solid material. The smaller the angle, the better the wettability; conversely, when the contact angle is greater than 90°, the liquid is said to be non-wettable to the surface of the solid material. The larger the angle, the worse the wettability.
  • the liquid is water, the material can be divided into hydrophilic and hydrophobic materials according to the size of the contact angle on the surface of the solid material.
  • Hydrophilic materials are more conducive to cell adhesion and spreading, while hydrophobic (or super-hydrophobic) environments are not conducive to cell adhesion. However, if the hydrophilicity of the material is too high, a hydration layer is easily formed on the surface of the material, which is also not conducive to the large-scale culture of adherent cells.
  • Three test materials were selected for testing. The materials were PET films of the same material and the porosities were measured to be 83%, 84%, and 76%, respectively. The PET films are the PET films mentioned in the present invention. The experimental results of the hydrophilicity and hydrophobicity of the three are shown in Figure 7.
  • the contact angles of the three selected cell culture PET film materials with porosities of 83%, 84%, and 76% are 55.75°, 39.5°, and 102.75°, respectively, from left to right in Figure 7.
  • the contact angle of the selected PET film is 39.5°, and the hydrophilicity is suitable.
  • the hydrophilicity of the PET film is not affected after high-pressure steam sterilization. Due to the breakage of trace ester bonds at high temperature, hydrophilic carboxyl and hydroxyl groups are generated, and the hydrophilicity is slightly improved, which has a beneficial effect on the attachment and growth of adherent cells.
  • PET film needs to be set to transparent and colorless.
  • Transmittance is the main property of PET film material. Generally, the transmittance is above 90%, which is conducive to observing the cell culture status under a microscope. In addition, it has the advantages of easy coloring, good processing fluidity, good rigidity and good chemical corrosion resistance.
  • the surface structure of the scaffold is also one of the indicators that affect the quality of cell culture.
  • the surface of the PET film has been hardened, that is, the surface layer of the part is hardened by an appropriate method while the core of the part still has strong toughness.
  • PET as a polymer film, has excellent optical properties and physical and mechanical properties, but due to the defects of the polymer itself, the surface hardness of most polymer films is low.
  • the additional surface hardening coating is thermally cured at high temperature on the surface of the material to increase the hardness of the material surface, thereby increasing the hardness of the fiber material to support a large number of cells growing on the membrane fiber.
  • the general process of hardening treatment is to use dibutyltin dilaurate as a curing agent, heat cure at 120°C for 3 hours, and obtain a PET surface hardening degree between 3H and 4H.
  • the surface hardening value is between 3H and 4H, which better supports cell adhesion.
  • the PET membrane of the present invention needs to be configured into a membrane column and a membrane mesh support structure in structure, and there are also certain requirements in parameter setting.
  • the specific requirements are as follows:
  • the hardening value of the film surface of the PET film after hardening treatment is between 3H and 4H;
  • the fibers inside the PET film are in a grid shape, and the fiber diameter is between 20-30 ⁇ m;
  • the tensile strength of the PET film is in the range of 3-6.5MPa
  • the PET film is hydrophilic
  • the PET film is transparent and colorless as a whole, and its light transmittance is greater than 90%.
  • the PET film screened out can be used as a carrier in cell and gene therapy to achieve application in cell culture.
  • this kind of PET film can be used for the cultivation of a variety of cells.
  • the adherent cells that the PET film can be used to cultivate include mammalian cells, somatic cells, stem cells, progenitor cells, mature cells, fibroblasts, skeletal tissue (bone and cartilage), myocardium and smooth muscle, liver, lung, kidney, breast skin, glial cells, endocrine cells, melanocytes and various tumor cells of people, mice, rats, pigs, cattle and monkeys, etc. These types of cells are all disclosed cells, and only examples are given in the present invention without specific restrictions.
  • mature cells may include nerve cells, cardiomyocytes, retinal cells and hepatocytes, etc.
  • stem cells may include mesenchymal stem cells (MSC), iPS cells, ES cells, Muse cells, embryonic cancer cells, embryonic germ stem cells, etc.
  • the present invention provides a corresponding method of use, and the specific working process is as follows:
  • the PET film is treated with alkali neutralization to remove bacterial endotoxins, sterilized to remove bacteria and mycoplasma, and mycoplasma and bacterial endotoxin tests are performed to avoid affecting the growth and quality of cells during cell culture.
  • Endotoxins are lipopolysaccharides in the cell wall of Gram-negative bacteria. Endotoxins are released only when the bacteria die and dissolve or the bacterial cells are destroyed by artificial methods. Alkaline solutions can chemically degrade endotoxins on glass, plastic and other polymer material vessels.
  • the specific alkali neutralization treatment is: 0.5M NaOH solution completely soaks the PET film for 2 hours, then ultrapure water is circulated and rinsed twice, the first rinse is 2 hours, and then the ultrapure water is replaced and rinsed for 4 hours; high temperature and high pressure are used to eliminate bacteria and mycoplasma.
  • the specific sterilization treatment is: put the PET film after alkali neutralization into a high-pressure steam sterilizer, the pressure is 100kPa, the temperature is 121°C, sterilize for 45 minutes and then dry. After the alkali neutralization and sterilization treatment, the PET film continues to be tested for mycoplasma and bacterial endotoxin.
  • the standard for mycoplasma detection is mycoplasma-free.
  • the PET film that has passed various parameter tests is placed in a well plate, and after adding an appropriate amount of culture medium, cells are inoculated.
  • the carrier is transferred to a new well plate every 3 hours, and the unattached cells are collected and counted, and the attachment rate is calculated until the attachment rate is close to 80%; the above-mentioned attached cells are continued to be cultured, and the culture medium is regularly replaced during the culture process.
  • Cell sampling is performed every 24 hours, and the cells removed from the PET film are digested and lysed, and the cell density is detected to draw a growth curve.
  • the PET film when used for cell culture, several small long sampling strips can be cut from the PET film and placed at different positions of the inner layer, middle layer and outer layer of the membrane column. More positions inside the PET film can be sampled during in vitro cell experiments. The setting of the sampling strips can facilitate experimental monitoring and meet the needs of exploring various parameters and conditions and process development.
  • the corresponding container carrier can use a container body (container) known in the past.
  • the shape and size of the container body are not particularly limited.
  • a cell culture dish, a square bottle for cell culture or a spinner bottle can be used, and as a large-scale cell culture container body, a cell factory or a cell bioreactor can be used.
  • HEK293T cells are attached and cultured in a PET film as an example.
  • the specific description is as follows:
  • the preferred PET membrane was placed in a well plate, and after adding an appropriate amount of culture medium, HEK293T cells were inoculated.
  • the carrier was transferred to a new well plate every 3 hours, and the unattached cells were collected for counting.
  • Figure 8 After HEK293T cells were inoculated on the PET membrane, the cell attachment rate gradually increased with time: 3 hours after cell inoculation, more than 60% of the cells were attached; as time went on, the attachment rate further increased, and the attachment rate remained basically stable after 12 hours of inoculation, and the final attachment rate was close to 80%.
  • HEK293T cells have good adhesion to PET membranes under static conditions. If used in dynamic culture conditions of bioreactors, this adhesion rate can be further improved, making it suitable for industrial large-scale cell culture.

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Abstract

The present invention relates to the technical field of cell culture and processing, and provides a PET film for cell culture in cell and gene therapy, and the use thereof. The PET film is a long-scroll-like coiled film, and forms a cylindrical film column overall after being spirally coiled, wherein the structure of the PET film is a regular and criss-crossed film net support structure. The hardening value of the surface of the PET film is between 3H and 4H after a hardening treatment. Fibers in the PET film are in a grid shape, and the diameters of the fibers are in the range of 20-30 μm. Under drying conditions, the tensile strength of the PET film is in the range of 3-6.5 MPa. The PET film is hydrophilic, and the PET film is transparent and colorless overall and has a light transmittance greater than 90%.

Description

一种细胞和基因治疗中用于细胞培养的PET膜及应用A PET film for cell culture in cell and gene therapy and its application 技术领域Technical Field
本发明涉及细胞培养技术领域，具体涉及到一种细胞和基因治疗中用于细胞培养的PET膜及应用。The invention relates to the technical field of cell culture, and in particular to a PET film used for cell culture in cell and gene therapy and an application thereof.
背景技术Background technique
细胞治疗和基因治疗(CGT)可在分子层面通过基因表达、沉默或体外改造的手段来实现疗法升级或治疗罕见病。细胞培养技术广泛应用于生物学研究、疾病诊断、组织工程、药物筛选等技术领域，该技术也是实现CGT规模化生产的技术核心。用于3D细胞培养体系中的PET(Polyethyleneterephthalate)膜，是一种新型的细胞培养材料，为贴壁细胞的生长繁殖提供了一种三维立体环境。传统的2D细胞培养器皿，与体内细胞真实生长环境存在着较大差异，导致细胞生长特性及功能一定程度上发生改变，PET膜是一种与体内生理状态更加接近的实验材料，这种3D细胞培养体系更好地模拟了体内细胞生长的微环境，且容易实现规模放大。Cell therapy and gene therapy (CGT) can achieve therapy upgrades or treat rare diseases at the molecular level through gene expression, silencing or in vitro modification. Cell culture technology is widely used in biological research, disease diagnosis, tissue engineering, drug screening and other technical fields. This technology is also the technical core for realizing large-scale production of CGT. The PET (Polyethyleneterephthalate) membrane used in the 3D cell culture system is a new type of cell culture material that provides a three-dimensional environment for the growth and reproduction of adherent cells. Traditional 2D cell culture vessels are quite different from the actual growth environment of cells in the body, which causes cell growth characteristics and functions to change to a certain extent. PET membrane is an experimental material that is closer to the physiological state in the body. This 3D cell culture system better simulates the microenvironment of cell growth in the body and is easy to scale up.
现有3D细胞培养主要是无支架培养体系，即通过悬滴让细胞在重力的作用下自组装形成的微球体或微载体材料。然而，这些方法实验操作复杂度较高、装置耗材成本昂贵，人员需要额外的技术培训，需制备的细胞接种量较大。因此，在细胞培养领域需要一种简单高效的、性价比高、适用于贴壁细胞规模化培养的实验材料。Existing 3D cell culture is mainly a scaffold-free culture system, that is, microspheres or microcarrier materials are formed by self-assembly of cells under the action of gravity through hanging drops. However, these methods have high experimental operation complexity, expensive equipment and consumables, and personnel need additional technical training. The amount of cell inoculation required is large. Therefore, in the field of cell culture, there is a need for a simple, efficient, cost-effective experimental material suitable for large-scale culture of adherent cells.
发明内容Summary of the invention
针对现有技术所存在的不足，本发明目的在于提出一种细胞和基因治疗中用于细胞培养的PET膜，具体方案如下：In view of the shortcomings of the prior art, the present invention aims to provide a PET film for cell culture in cell and gene therapy, and the specific scheme is as follows:
一种细胞和基因治疗中用于细胞培养的PET膜，所述的PET膜为长轴式卷膜，螺旋卷曲后整体形成圆柱状的膜柱，其中，PET膜的结构为规则的纵横交错设置的膜网支架结构；A PET film for cell culture in cell and gene therapy, wherein the PET film is a long-axis rolled film, which is spirally rolled to form a cylindrical film column as a whole, wherein the structure of the PET film is a regular crisscrossed membrane network support structure;
该PET膜的膜表面经过硬化处理之后的硬化值在3H-4H之间；The hardening value of the film surface of the PET film after hardening treatment is between 3H and 4H;
该PET膜内部的纤维呈网格状，且纤维直径在20-30μm范围之间；The fibers inside the PET film are in a grid shape, and the fiber diameter is between 20-30 μm;
在干燥条件下，该PET膜的拉伸强度在3-6.5MPa范围之间；Under dry conditions, the tensile strength of the PET film is in the range of 3-6.5MPa;
该PET膜具有亲水性；The PET film is hydrophilic;
该PET膜整体呈透明无色，透光率大于90％。The PET film is transparent and colorless as a whole, and its light transmittance is greater than 90%.
致密结构的PET膜经螺旋卷曲后形成的圆柱体多层膜网结构，对细胞有良好的拦截作用，并且在初期进行细胞接种时，浓稠的细胞悬液可以穿过膜的层层孔隙，另细胞均匀地固定于立体的3D培养系统，实现均一分布；The cylindrical multilayer membrane structure formed by spiral curling of the densely structured PET membrane has a good interception effect on cells. In the initial stage of cell inoculation, the thick cell suspension can pass through the pores of the membrane, and the cells are evenly fixed in the three-dimensional 3D culture system to achieve uniform distribution.
PET膜的纤维呈网格状多孔隙的结构，传质阻力较小，利于液体在孔隙间的穿梭流动，培养基液流可以顺利地穿过膜网，没有死角地与细胞进行充分接触，使细胞全方位地充分接触培养基，在扩增阶段吸收的营养物质均衡，故细胞的生长状态更均匀，细胞存活率更高。The fibers of the PET membrane have a grid-like porous structure with low mass transfer resistance, which is conducive to the shuttling flow of liquid between the pores. The culture medium flow can smoothly pass through the membrane network and fully contact the cells without dead corners, so that the cells can fully contact the culture medium in all directions and absorb a balanced amount of nutrients during the expansion stage, so the cell growth state is more uniform and the cell survival rate is higher.
进一步的，所述的PET膜可用于培养的贴壁细胞包括哺乳动物细胞，体细胞，干细胞，祖细胞，成熟细胞，成纤维细胞，骨骼组织，心肌与平滑肌、肝、肺、肾、乳腺皮肤、神经胶质细胞，内分泌细胞，黑色素细胞以及各种肿瘤细胞。Furthermore, the PET membrane can be used to culture adherent cells including mammalian cells, somatic cells, stem cells, progenitor cells, mature cells, fibroblasts, bone tissue, myocardium and smooth muscle, liver, lung, kidney, breast skin, glial cells, endocrine cells, melanocytes and various tumor cells.
进一步的，成熟细胞可包括神经细胞、心肌细胞、视网膜细胞和肝细胞；Further, mature cells may include neural cells, cardiomyocytes, retinal cells, and hepatocytes;
干细胞可包括间充质干细胞、iPS细胞、ES细胞、Muse细胞、胚胎癌症细胞、胚胎生殖干细胞。Stem cells may include mesenchymal stem cells, iPS cells, ES cells, Muse cells, embryonic cancer cells, and embryonic germ stem cells.
进一步的，所述的PET膜进行细胞贴附、培养的工作过程为：Furthermore, the working process of the PET film for cell attachment and culture is as follows:
将通过各种参数测试后的PET膜置于孔板中，加入适量培养基后，接种细胞，每隔3小时将载体转移至新的孔板，并收集未贴附的细胞进行计数，计算得到贴附率，直至贴附率接近80％；The PET membrane that has passed various parameter tests is placed in a well plate, and after adding an appropriate amount of culture medium, cells are inoculated. The carrier is transferred to a new well plate every 3 hours, and the unattached cells are collected and counted to calculate the attachment rate until the attachment rate is close to 80%;
继续对上述完成贴附的细胞进行培养，培养过程中定时更换培养基，每隔24小时进行细胞取样，对从PET膜上取下的细胞进行消化裂解后，进行细胞密度的检测，绘制生长曲线。Continue to culture the attached cells, change the culture medium regularly during the culture process, take cell samples every 24 hours, digest and lyse the cells removed from the PET membrane, detect the cell density, and draw a growth curve.
进一步的，在PET膜进行细胞贴附之前，对所述PET膜进行碱中和处理、灭菌处理以及支原体检测和细菌内毒素检测；Furthermore, before the cells are attached to the PET film, the PET film is subjected to alkali neutralization treatment, sterilization treatment, and mycoplasma detection and bacterial endotoxin detection;
碱中和处理具体为：0.5M的NaOH溶液完全浸泡PET膜2h后，超纯水循环冲洗2遍，第一次冲洗2h,更换超纯水后再进行二次冲洗4h；The specific alkaline neutralization treatment is as follows: the PET film is completely immersed in a 0.5M NaOH solution for 2 hours, and then rinsed twice with ultrapure water. The first rinse is 2 hours, and then the ultrapure water is replaced and rinsed for a second time for 4 hours.
灭菌处理具体为：将经过碱中和处理后的PET膜放入高压蒸汽灭菌锅中，压力100kPa，温度121℃，灭菌45min后晾干；The sterilization treatment is as follows: the PET film after alkali neutralization treatment is placed in a high-pressure steam sterilizer at a pressure of 100 kPa and a temperature of 121°C, sterilized for 45 minutes and then dried;
支原体检测的标准为无支原体，细菌内毒素检测的标准为内毒素的含量＜0.25EU/件。The standard for mycoplasma testing is the absence of mycoplasma, and the standard for bacterial endotoxin testing is the endotoxin content of <0.25EU/piece.
进一步的，所述PET膜进行细胞培养时，对应的容器载体可采用细胞培养皿、细胞培养用方瓶或转瓶、细胞工厂、细胞生物反应器。Furthermore, when the PET film is used for cell culture, the corresponding container carrier can be a cell culture dish, a square bottle or a rotating bottle for cell culture, a cell factory, or a cell bioreactor.
进一步的，所述PET膜进行细胞培养时，可将PET膜裁剪出若干个小型长条取样条，所述长条取样条放置在所述膜柱的内层、中间层以及外层的不同位置。Furthermore, when the PET film is used for cell culture, the PET film can be cut into a plurality of small long sampling strips, and the long sampling strips are placed at different positions of the inner layer, the middle layer and the outer layer of the membrane column.
所述细胞和基因治疗中用于细胞培养的PET膜作为载体，在细胞培养中的应用。The PET film used for cell culture in cell and gene therapy is used as a carrier in cell culture.
与现有技术相比，本发明的有益效果如下：Compared with the prior art, the present invention has the following beneficial effects:
(1)现有贴壁细胞培养主要有2D培养和3D培养两种形式，2D培养方法为平面法培养，其培养面的面积限制了贴壁细胞的扩增倍数，产量较少，不能满足市场大规模生产的需求。相较于现有技术，本发明的PET膜的多层致密的膜网结构所构建的3D培养体系给细胞提供了几何倍数的立体生长空间，有效避免了培养过程中出现接触抑制而降低扩增速率的问题，细胞扩增量远远高于普通培养模式，适用于大规模生产和工艺放大。(1) Existing adherent cell culture mainly includes two forms: 2D culture and 3D culture. The 2D culture method is a planar culture method, and the area of its culture surface limits the expansion multiples of adherent cells, resulting in a low yield and failing to meet the market's demand for large-scale production. Compared with the prior art, the 3D culture system constructed by the multi-layer dense membrane network structure of the PET film of the present invention provides cells with a geometric multiple three-dimensional growth space, effectively avoiding the problem of contact inhibition during the culture process and reducing the expansion rate. The cell expansion amount is much higher than that of the ordinary culture mode, and is suitable for large-scale production and process amplification.
(2)本发明解决了现有技术中无法针对贴壁细胞进行大规模3D培养的问题，PET膜的支架结构所构建的3D细胞培养系统很好地模仿体内细胞生长的微你环境，对于研究细胞生理状态和活动有更科学的借鉴意义。(2) The present invention solves the problem in the prior art that adherent cells cannot be cultured on a large scale in 3D. The 3D cell culture system constructed by the scaffold structure of the PET membrane well mimics the microenvironment of cell growth in vivo, which has a more scientific reference significance for studying the physiological state and activity of cells.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为PET分子结构示意图。Figure 1 is a schematic diagram of the molecular structure of PET.
图2为适用于大规模细胞培养的PET膜的结构示意图。FIG. 2 is a schematic diagram of the structure of a PET membrane suitable for large-scale cell culture.
图3为PET膜支架结构及其对细胞贴附作用示意图。FIG. 3 is a schematic diagram of the PET membrane scaffold structure and its effect on cell attachment.
图4为PET膜显微结构示意图。FIG4 is a schematic diagram of the microstructure of the PET film.
图5为电镜下PET膜的孔隙率直观图。Figure 5 is a direct view of the porosity of the PET film under an electron microscope.
图6为PET膜的拉伸曲线图。FIG. 6 is a tensile curve diagram of the PET film.
图7为材料亲疏水性测试示意图。FIG. 7 is a schematic diagram of a material hydrophilicity and hydrophobicity test.
图8为HEK293T细胞在PET膜上的贴附情况示意图。FIG. 8 is a schematic diagram showing the adhesion of HEK293T cells to the PET membrane.
实施方式Implementation
下面结合实施例及附图对本发明作进一步的详细说明，但本发明的实施方式不仅限于此。The present invention will be further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.
现有技术中，PET全称是聚对苯二甲酸乙二醇酯，它是由对苯二甲酸与乙二醇缩合聚合反应而制得的，其电性能较好，抗蠕变性、耐疲劳性、耐摩擦性、材料稳定性都较好。具体阐述如下：In the prior art, PET stands for polyethylene terephthalate, which is produced by the condensation polymerization of terephthalic acid and ethylene glycol. It has good electrical properties, creep resistance, fatigue resistance, friction resistance, and material stability. The specific description is as follows:
1.有良好的力学性能，冲击强度是其他材质薄膜的3-5倍，耐折性好。1. It has good mechanical properties, the impact strength is 3-5 times that of other materials, and it has good folding resistance.
2.耐油脂类、耐稀酸、稀碱，耐大多数溶剂。和实验试剂安全性接触，维持膜的原有特性。2. Resistant to oils, fats, dilute acids, alkalis, and most solvents. It can safely contact with experimental reagents and maintain the original properties of the membrane.
3.PET有酯键，高、低温环境下对其机械性能影响很小，使用温度达-100至120℃。可耐受高温高压灭菌，且高压蒸汽灭菌后材料的亲水性得到了小幅度改善提升，原因是高温下微量的酯键断裂产生了亲水的羧基和羟基。3. PET has ester bonds, and its mechanical properties are little affected by high and low temperature environments, and the operating temperature is -100 to 120°C. It can withstand high temperature and high pressure sterilization, and the hydrophilicity of the material has been slightly improved after high pressure steam sterilization, because the trace amount of ester bonds are broken at high temperature to produce hydrophilic carboxyl and hydroxyl groups.
4.气体和水蒸汽渗透率低，有优良的阻气、水、油及异味性能。4. The gas and water vapor permeability is low, and it has excellent gas, water, oil and odor barrier properties.
5.透明度高，可耐紫外线杀菌，透光性及光泽性好。5. High transparency, resistant to UV sterilization, good light transmittance and gloss.
6.无毒、无味，卫生安全性好，适用于医疗包装和器材设备。6. Non-toxic, odorless, hygienic and safe, suitable for medical packaging and equipment.
而且，生产PET膜所用的乙二醇价格便宜，就原材料制造成本而言，PET材料是工程塑料中价格最低的，具有很高的性价比。Moreover, the ethylene glycol used to produce PET film is cheap. In terms of raw material manufacturing cost, PET material is the lowest price among engineering plastics and has a high cost-effectiveness.
基于上述内容，本发明提出一种细胞和基因治疗中用于细胞培养的PET膜，该PET膜具体为一种用于贴壁细胞3D培养的实验材料，材质为PET(聚对苯二甲酸乙二醇酯)膜，化学式为(C10H8O4)n，分子结构图如图1。Based on the above content, the present invention proposes a PET film for cell culture in cell and gene therapy. The PET film is specifically an experimental material for 3D culture of adherent cells. The material is PET (polyethylene terephthalate) film, the chemical formula is (C10H8O4)n, and the molecular structure diagram is shown in Figure 1.
具体来说，本发明的PET膜的在整体外观构造上来看，为长轴式卷膜，螺旋卷曲后整体形成圆柱状的膜柱(如图2)，在使用时，可按细胞培养容器或载体的规格灵活适配裁剪。Specifically, the PET film of the present invention is a long-axis rolled film in terms of overall appearance and structure, which is spirally rolled to form a cylindrical film column as a whole (as shown in FIG. 2 ). When used, it can be flexibly adapted and cut according to the specifications of the cell culture container or carrier.
支架是常用的培养基质材料，它可以提供物理支撑，细胞可以进入支架内部进行生长和行使功能(如图3)。因此，本发明的PET膜的结构从内部构造来看，为规则的纵横交错设置的膜网支架结构，利用膜网支架结构对细胞的拦截作用从而实现贴壁细胞的3D培养，因此，PET膜内部的膜网支架结构具有生物安全性的同时，稳定性和力学性也能相对较好。A scaffold is a commonly used culture matrix material that can provide physical support, and cells can enter the scaffold to grow and function (as shown in FIG3). Therefore, the structure of the PET film of the present invention is a regular crisscrossed membrane network scaffold structure from the internal structure, and the membrane network scaffold structure is used to intercept cells to achieve 3D culture of adherent cells. Therefore, the membrane network scaffold structure inside the PET film has biological safety, and the stability and mechanical properties are relatively good.
当然，支架的表面结构、孔隙率和支架的力学性能等也都会影响细胞的迁移行为和生长状态。支架同样可以进行功能化赋予不同的性能，进一步探究细胞的3D行为。Of course, the surface structure, porosity and mechanical properties of the scaffold will also affect the migration behavior and growth state of cells. Scaffolds can also be functionalized to give them different properties to further explore the 3D behavior of cells.
因此，本发明根据贴壁细胞的生长特性，对各种PET材质的膜结构展开一系列试验测试，确定合适的参数，筛选出最适合细胞大规模贴壁培养的PET膜材料，这种PET膜便是本发明的所要保护的内容。Therefore, according to the growth characteristics of adherent cells, the present invention conducts a series of experimental tests on membrane structures of various PET materials, determines appropriate parameters, and screens out the PET membrane material that is most suitable for large-scale cell adherent culture. This PET membrane is the content to be protected by the present invention.
首先，PET膜需要存在一定的空隙用于细胞和培养基的分布，将PET膜材料固定在样品台上进行喷金处理，随后用扫描电子显微镜进行观察，结果如图4所示。从PET膜的显微结构来看，可见PET膜的纤维呈网格状均匀地分布在视野中，形成了多空隙的结构，传质阻力较小，有利于培养基的流动。纤维的交叉处有熔融的痕迹，采用熔喷的方法生产而成，其中少有热熔的痕迹，纤维之间存在膜状结构，因此通过粘合剂或交联剂来实现纤维间的连接。First, the PET film needs to have certain gaps for the distribution of cells and culture medium. The PET film material is fixed on the sample stage for gold spraying, and then observed with a scanning electron microscope. The results are shown in Figure 4. From the microstructure of the PET film, it can be seen that the fibers of the PET film are evenly distributed in the field of view in a grid shape, forming a multi-void structure with low mass transfer resistance, which is conducive to the flow of culture medium. There are traces of melting at the intersection of the fibers, which are produced by melt-blowing method. There are few traces of hot melting. There is a membrane structure between the fibers, so the connection between the fibers is achieved by adhesives or cross-linking agents.
其次，PET膜的纤维直径和孔隙率是影响细胞与培养表面接触概率的关键性指标，接近贴壁细胞(20μm-50μm)的纤维直径，有利于细胞贴附和铺展。其中，本发明的PET膜的纤维直径在20-30μm范围之间，利于细胞贴壁着床，为了防止孔隙太大，细胞难以贴附于材料生长，导致细胞聚团或死亡等较为严重的情况出现，本发明进行了纤维直径测试实验，筛选多个具有不同纤维直径的PET材料后，选择具有合适的纤维直径的PET膜材料。具体方法如下：Secondly, the fiber diameter and porosity of the PET film are key indicators that affect the probability of contact between cells and the culture surface. A fiber diameter close to that of adherent cells (20 μm-50 μm) is conducive to cell attachment and spreading. Among them, the fiber diameter of the PET film of the present invention is between 20-30 μm, which is conducive to cell attachment and implantation. In order to prevent the pores from being too large, making it difficult for cells to attach to the material for growth, leading to more serious situations such as cell aggregation or death, the present invention conducts a fiber diameter test experiment, and after screening multiple PET materials with different fiber diameters, selects a PET film material with a suitable fiber diameter. The specific method is as follows:
将不同PET膜的电镜照片导入ImageJ软件中，需要说明的是，该软件为纤维直径测试实验中自带的现有软件，不涉及到改进。选定表层的纤维后，计算所选区域占整张照片的面积，可以得到材料的孔隙率。处理结果如图5从左至右的a、b、c三张小图所示，经计算所筛选的三种PET膜材料孔隙率分别为83％、84％、76％。优选的，孔隙率为84％的PET膜，细胞贴附性强，细胞的贴壁率最高。The electron microscope photos of different PET films are imported into ImageJ software. It should be noted that this software is the existing software that comes with the fiber diameter test experiment and does not involve improvement. After selecting the surface fibers, the area of the selected area in the entire photo is calculated to obtain the porosity of the material. The processing results are shown in the three small pictures a, b, and c from left to right in Figure 5. The porosities of the three PET film materials selected by calculation are 83%, 84%, and 76%, respectively. Preferably, the PET film with a porosity of 84% has strong cell adhesion and the highest cell adhesion rate.
再其次，PET膜的力学性能也是影响的指标之一，本发明对PET膜进行了拉伸强度测试，具体方法如下：Secondly, the mechanical properties of the PET film are also one of the influencing indicators. The present invention conducts a tensile strength test on the PET film, and the specific method is as follows:
将PET膜剪成条状样品，通过厚度和宽度测出横截面积，然后将样品的上下端固定于拉伸仪上，测得样品的拉伸曲线。如图6所示，由于材料的纤维排列方向和分布密度是随机的，因此每份样品的抗拉伸能力都存在差异，湿的PET膜较干的PET膜的拉伸强度稍稍略低，原因是PET膜中的粘合剂遇水后粘合作用下降，导致PET膜变松散。优选的，PET膜的拉伸强度在3-6.5MPa之间。此时的PET膜有良好的力学性能，耐折性、抗蠕变性、耐疲劳性、尺寸稳定性都很好，最适合用于大规模细胞培养。PET film is cut into strip samples, the cross-sectional area is measured by thickness and width, and then the upper and lower ends of the sample are fixed on a tensile tester to measure the tensile curve of the sample. As shown in Figure 6, since the fiber arrangement direction and distribution density of the material are random, there are differences in the tensile strength of each sample, and the tensile strength of the wet PET film is slightly lower than that of the dry PET film, because the adhesive in the PET film decreases after the adhesive is exposed to water, causing the PET film to become loose. Preferably, the tensile strength of the PET film is between 3-6.5MPa. The PET film at this time has good mechanical properties, and folding resistance, creep resistance, fatigue resistance, and dimensional stability are all very good, which is most suitable for large-scale cell culture.
再其次，固体材料表面的润湿性是固体材料表面的重要性质，表面润湿性能取决于材料表面的化学成分和微观的几何结构。当液滴滴在固体材料水平表面上时，液滴可能铺展于材料表面或形成一定角度的液滴停在表面，在固、液、气三相交界处，自固-液界面经过液体内部到气-液界面之间的夹角称为接触角(θ)。通常以接触角来衡量某种固体材料表面的润湿性。习惯上，将θ＝90°作为材料表面润湿与否的标准。当接触角小于90°时，称该液体可润湿固体材料，角度越小，润湿性越好；反之，当接触角大于90°时，称该液体不可润湿固体材料表面，角度越大，润湿性越差。若该液体是水，就可以根据固体材料表面接触角大小，将材料分为亲水性材料和疏水性材料。Secondly, the wettability of the surface of solid materials is an important property of the surface of solid materials. The surface wettability depends on the chemical composition and microscopic geometric structure of the material surface. When a droplet falls on the horizontal surface of a solid material, the droplet may spread on the surface of the material or form a droplet at a certain angle and stop on the surface. At the junction of solid, liquid and gas, the angle from the solid-liquid interface through the liquid to the gas-liquid interface is called the contact angle (θ). The contact angle is usually used to measure the wettability of a certain solid material surface. Conventionally, θ = 90° is used as the standard for whether the material surface is wettable or not. When the contact angle is less than 90°, the liquid is said to be wettable to the solid material. The smaller the angle, the better the wettability; conversely, when the contact angle is greater than 90°, the liquid is said to be non-wettable to the surface of the solid material. The larger the angle, the worse the wettability. If the liquid is water, the material can be divided into hydrophilic and hydrophobic materials according to the size of the contact angle on the surface of the solid material.
亲水材料更利于细胞黏附与铺展，疏水(或超疏水)环境不利于细胞的黏附。但如果材料的亲水性过高，则容易在材料的表面形成水化层，同样不利于贴壁细胞的大规模培养。选用了三种测试材料进行测试，材料为相同材质且孔隙率经测定分别为83％、84％、76％的PET膜，PET膜为本发明中提到的PET膜，三者亲疏水性的实验结果如图7所示。所选择的三种孔隙率分别为83％、84％、76％的细胞培养PET膜材料从图7中从左至右的顺序的接触角分别为55.75°、39.5°、102.75°。优选的，所选的PET膜接触角为39.5°，亲水性合适，而且经实验验证，高压蒸汽灭菌后PET膜的亲水性不仅不受影响，由于高温下微量的酯键断裂产生了亲水的羧基和羟基，亲水性能略有提高，对贴壁细胞的贴附和生长均属有益的影响。Hydrophilic materials are more conducive to cell adhesion and spreading, while hydrophobic (or super-hydrophobic) environments are not conducive to cell adhesion. However, if the hydrophilicity of the material is too high, a hydration layer is easily formed on the surface of the material, which is also not conducive to the large-scale culture of adherent cells. Three test materials were selected for testing. The materials were PET films of the same material and the porosities were measured to be 83%, 84%, and 76%, respectively. The PET films are the PET films mentioned in the present invention. The experimental results of the hydrophilicity and hydrophobicity of the three are shown in Figure 7. The contact angles of the three selected cell culture PET film materials with porosities of 83%, 84%, and 76% are 55.75°, 39.5°, and 102.75°, respectively, from left to right in Figure 7. Preferably, the contact angle of the selected PET film is 39.5°, and the hydrophilicity is suitable. Moreover, it has been verified by experiments that the hydrophilicity of the PET film is not affected after high-pressure steam sterilization. Due to the breakage of trace ester bonds at high temperature, hydrophilic carboxyl and hydroxyl groups are generated, and the hydrophilicity is slightly improved, which has a beneficial effect on the attachment and growth of adherent cells.
再其次，PET膜的材质特点也是影响细胞培养质量的指标之一，PET膜需要设置为透明无色的，透光率是PET膜材料的主要性质，一般透光率在90％以上，利于在显微镜下观察细胞培养状态，另外其具有易着色，加工流动性好，刚性好及耐化学腐蚀性好等优点。Secondly, the material characteristics of PET film are also one of the indicators that affect the quality of cell culture. PET film needs to be set to transparent and colorless. Transmittance is the main property of PET film material. Generally, the transmittance is above 90%, which is conducive to observing the cell culture status under a microscope. In addition, it has the advantages of easy coloring, good processing fluidity, good rigidity and good chemical corrosion resistance.
再其次，支架的表面结构也是影响细胞培养质量的指标之一，本发明中，对PET膜表面经过了硬化处理，即通过适当的方法使零件的表层硬化而零件的心部仍然具有强韧性的处理，PET作为高分子薄膜，具有优秀的光学性能和物理机械性能，但由于高分子自身的缺陷，大多数高分子薄膜表面硬度较低，通过在材料表面高温热固化附加的表面硬化涂层以提高材料表面的硬度，增加纤维材料的硬度以支撑膜纤维上数量众多贴壁生长的细胞。Secondly, the surface structure of the scaffold is also one of the indicators that affect the quality of cell culture. In the present invention, the surface of the PET film has been hardened, that is, the surface layer of the part is hardened by an appropriate method while the core of the part still has strong toughness. PET, as a polymer film, has excellent optical properties and physical and mechanical properties, but due to the defects of the polymer itself, the surface hardness of most polymer films is low. The additional surface hardening coating is thermally cured at high temperature on the surface of the material to increase the hardness of the material surface, thereby increasing the hardness of the fiber material to support a large number of cells growing on the membrane fiber.
硬化处理的大致过程为采用二月桂酸二丁基锡作为固化剂，在120℃下热固化3h，可得到PET表面硬化度在3H-4H之间。PET膜表面硬化越好，材料支撑性越强，耐磨性越强。优选的，表面的硬化值在3H-4H之间，较好地支撑细胞粘附。The general process of hardening treatment is to use dibutyltin dilaurate as a curing agent, heat cure at 120°C for 3 hours, and obtain a PET surface hardening degree between 3H and 4H. The better the surface hardening of the PET film, the stronger the material support and the stronger the wear resistance. Preferably, the surface hardening value is between 3H and 4H, which better supports cell adhesion.
基于上述内容，综述来说，为实现细胞大规模贴壁培养，本发明的PET膜除了在结构上需要设置成膜柱以及膜网支架结构，在参数设置上，也还有一定的要求，具体要求如下：Based on the above content, in summary, in order to achieve large-scale cell adherence culture, the PET membrane of the present invention needs to be configured into a membrane column and a membrane mesh support structure in structure, and there are also certain requirements in parameter setting. The specific requirements are as follows:
该PET膜的膜表面经过硬化处理之后的硬化值在3H-4H之间；The hardening value of the film surface of the PET film after hardening treatment is between 3H and 4H;
该PET膜内部的纤维呈网格状，且纤维直径在20-30μm范围之间；The fibers inside the PET film are in a grid shape, and the fiber diameter is between 20-30 μm;
在干燥条件下，该PET膜的拉伸强度在3-6.5MPa范围之间；Under dry conditions, the tensile strength of the PET film is in the range of 3-6.5MPa;
该PET膜具有亲水性；The PET film is hydrophilic;
该PET膜整体呈透明无色，透光率大于90％。The PET film is transparent and colorless as a whole, and its light transmittance is greater than 90%.
筛选出来的PET膜，可在细胞和基因治疗中作为载体，实现在细胞培养中的应用。实际上，该种PET膜可用于对多种细胞的培养，具体来说，PET膜可用于培养的贴壁细胞包括人、小鼠、大鼠、猪、牛和猴等的哺乳动物细胞，体细胞，干细胞，祖细胞，成熟细胞，成纤维细胞，骨骼组织(骨及软骨)，心肌与平滑肌、肝、肺、肾、乳腺皮肤、神经胶质细胞，内分泌细胞，黑色素细胞以及各种肿瘤细胞。这些种类的细胞均为已公开的细胞，本发明中只进行举例，不做具体限制。其中，比如成熟细胞可包括神经细胞、心肌细胞、视网膜细胞和肝细胞等，干细胞可包括间充质干细胞(MSC)、iPS细胞、ES细胞、Muse细胞、胚胎癌症细胞、胚胎生殖干细胞等。The PET film screened out can be used as a carrier in cell and gene therapy to achieve application in cell culture. In fact, this kind of PET film can be used for the cultivation of a variety of cells. Specifically, the adherent cells that the PET film can be used to cultivate include mammalian cells, somatic cells, stem cells, progenitor cells, mature cells, fibroblasts, skeletal tissue (bone and cartilage), myocardium and smooth muscle, liver, lung, kidney, breast skin, glial cells, endocrine cells, melanocytes and various tumor cells of people, mice, rats, pigs, cattle and monkeys, etc. These types of cells are all disclosed cells, and only examples are given in the present invention without specific restrictions. Wherein, for example, mature cells may include nerve cells, cardiomyocytes, retinal cells and hepatocytes, etc., and stem cells may include mesenchymal stem cells (MSC), iPS cells, ES cells, Muse cells, embryonic cancer cells, embryonic germ stem cells, etc.
在PET膜上进行细胞贴附、培养时，本发明对应提出了使用方法，具体工作过程如下：When cells are attached and cultured on the PET film, the present invention provides a corresponding method of use, and the specific working process is as follows:
首先，在PET膜进行细胞贴附之前，对PET膜进行碱中和处理以除去细菌内毒素、灭菌处理以除去细菌和支原体，并进行支原体检测和细菌内毒素检测，避免在细胞培养过程中影响细胞的生长以及质量。内毒素是革兰氏阴性细菌细胞壁中的脂多糖。内毒素只有当细菌死亡溶解或用人工方法破坏菌细胞后才释放出来，碱溶液可化学降解玻璃、塑料和其它高分子材料器皿上的内毒素，碱中和处理具体为：0.5M的NaOH溶液完全浸泡PET膜2h后，超纯水循环冲洗2遍，第一次冲洗2h,更换超纯水后再进行二次冲洗4h；利用高温高压消除细菌和支原体，灭菌处理具体为：将经过碱中和处理后的PET膜放入高压蒸汽灭菌锅中，压力100kPa，温度121℃，灭菌45min后晾干。进行完碱中和处理、灭菌处理的PET膜继续进行支原体检测和细菌内毒素检测，支原体检测的标准为无支原体，细胞培养物被支原体污染后会出现在各种比如生长减慢的问题；根据《中华人民共和国药典》2020版，通过鲎试剂与细菌内毒素产生凝集反应的原理，以判断供试品中内毒素限量是否符合规定。合格标准：产品细菌内毒素含量＜0.25EU/件，细胞培养物被内毒素污染后会出现在各种比如改变细胞外形的问题。当两个检测的结果为合格后，该PET膜可正常使用。First, before the cell is attached to the PET film, the PET film is treated with alkali neutralization to remove bacterial endotoxins, sterilized to remove bacteria and mycoplasma, and mycoplasma and bacterial endotoxin tests are performed to avoid affecting the growth and quality of cells during cell culture. Endotoxins are lipopolysaccharides in the cell wall of Gram-negative bacteria. Endotoxins are released only when the bacteria die and dissolve or the bacterial cells are destroyed by artificial methods. Alkaline solutions can chemically degrade endotoxins on glass, plastic and other polymer material vessels. The specific alkali neutralization treatment is: 0.5M NaOH solution completely soaks the PET film for 2 hours, then ultrapure water is circulated and rinsed twice, the first rinse is 2 hours, and then the ultrapure water is replaced and rinsed for 4 hours; high temperature and high pressure are used to eliminate bacteria and mycoplasma. The specific sterilization treatment is: put the PET film after alkali neutralization into a high-pressure steam sterilizer, the pressure is 100kPa, the temperature is 121℃, sterilize for 45 minutes and then dry. After the alkali neutralization and sterilization treatment, the PET film continues to be tested for mycoplasma and bacterial endotoxin. The standard for mycoplasma detection is mycoplasma-free. After the cell culture is contaminated with mycoplasma, various problems such as slowed growth will occur. According to the 2020 edition of the "Pharmacopoeia of the People's Republic of China", the principle of agglutination reaction between horseshoe crab reagent and bacterial endotoxin is used to determine whether the endotoxin limit in the test sample meets the regulations. Qualification standard: The bacterial endotoxin content of the product is less than 0.25EU/piece. After the cell culture is contaminated with endotoxin, various problems such as changes in cell appearance will occur. When the results of the two tests are qualified, the PET film can be used normally.
将通过各种参数测试后的PET膜置于孔板中，加入适量培养基后，接种细胞，每隔3小时将载体转移至新的孔板，并收集未贴附的细胞进行计数，计算得到贴附率，直至贴附率接近80％；继续对上述完成贴附的细胞进行培养，培养过程中定时更换培养基，每隔24小时进行细胞取样，对从PET膜上取下的细胞进行消化裂解后，进行细胞密度的检测，绘制生长曲线。The PET film that has passed various parameter tests is placed in a well plate, and after adding an appropriate amount of culture medium, cells are inoculated. The carrier is transferred to a new well plate every 3 hours, and the unattached cells are collected and counted, and the attachment rate is calculated until the attachment rate is close to 80%; the above-mentioned attached cells are continued to be cultured, and the culture medium is regularly replaced during the culture process. Cell sampling is performed every 24 hours, and the cells removed from the PET film are digested and lysed, and the cell density is detected to draw a growth curve.
需要说明的是，PET膜进行细胞培养时，可将PET膜裁剪出若干个小型长条取样条，长条取样条放置在膜柱的内层、中间层以及外层的不同位置，可以对体外细胞实验时PET膜内部更多的位置进行取样，取样条的设置可以方便实验监测，满足各参数和条件的摸索，工艺开发的需求。It should be noted that when the PET film is used for cell culture, several small long sampling strips can be cut from the PET film and placed at different positions of the inner layer, middle layer and outer layer of the membrane column. More positions inside the PET film can be sampled during in vitro cell experiments. The setting of the sampling strips can facilitate experimental monitoring and meet the needs of exploring various parameters and conditions and process development.
PET膜进行细胞培养时，对应的容器载体可使用以往公知的容器主体(容器)。容器主体的形状和尺寸没有特别限定。作为容器主体，可采用细胞培养皿、细胞培养用方瓶或转瓶，作为大规模细胞培养容器主体，可采用细胞工厂、细胞生物反应器。When the PET film is used for cell culture, the corresponding container carrier can use a container body (container) known in the past. The shape and size of the container body are not particularly limited. As the container body, a cell culture dish, a square bottle for cell culture or a spinner bottle can be used, and as a large-scale cell culture container body, a cell factory or a cell bioreactor can be used.
结合上述内容，本实施例中，以HEK293T细胞在PET膜中进行贴附、培养进行举例说明。具体阐述如下：In combination with the above content, in this embodiment, HEK293T cells are attached and cultured in a PET film as an example. The specific description is as follows:
将经过各种参数筛选测试后，优选的PET膜置于孔板中，加入适量培养基后，接种HEK293T细胞，每隔3小时将载体转移至新的孔板，并收集未贴附的细胞进行计数。结果如图8所示，HEK293T细胞接种到PET膜上后，随着时间延长，细胞贴附率逐渐升高：细胞接种后3小时，即有60％以上的细胞完成贴附；随着时间延长，贴附率进一步提高，接种12小时后贴附率基本保持稳定，最终贴附率接近80％。After various parameter screening tests, the preferred PET membrane was placed in a well plate, and after adding an appropriate amount of culture medium, HEK293T cells were inoculated. The carrier was transferred to a new well plate every 3 hours, and the unattached cells were collected for counting. The results are shown in Figure 8. After HEK293T cells were inoculated on the PET membrane, the cell attachment rate gradually increased with time: 3 hours after cell inoculation, more than 60% of the cells were attached; as time went on, the attachment rate further increased, and the attachment rate remained basically stable after 12 hours of inoculation, and the final attachment rate was close to 80%.
此时可知，在静态条件下HEK293T细胞在PET膜上的贴附能力良好。如果用于生物反应器的动态培养条件，此贴附率可进一步得到提高，适合工业化大规模细胞培养。It can be seen that HEK293T cells have good adhesion to PET membranes under static conditions. If used in dynamic culture conditions of bioreactors, this adhesion rate can be further improved, making it suitable for industrial large-scale cell culture.
继续对上述完成贴附的HEK293T细胞进行培养，培养过程中注意及时更换培养基，及跟踪各培养参数的变化，同时每隔24小时进行细胞取样；将优选的PET膜上的细胞进行消化裂解后，多次实验进行细胞密度的检测确定，绘制细胞生长曲线。最终实验结果如图8所示，从图中可以看到细胞在优选的PET膜上能够正常进行扩增和生长，生长曲线良好，效果优于其他参数的PET膜材料。Continue to culture the HEK293T cells that have been attached as described above, pay attention to timely replacement of the culture medium during the culture process, track the changes in various culture parameters, and sample cells every 24 hours; after digesting and lysing the cells on the preferred PET film, perform multiple experiments to detect and determine the cell density, and draw a cell growth curve. The final experimental results are shown in Figure 8, from which it can be seen that the cells can be normally amplified and grown on the preferred PET film, the growth curve is good, and the effect is better than the PET film materials with other parameters.
以上所述仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims (8)

  1. 一种细胞和基因治疗中用于细胞培养的PET膜，其特征在于，所述的PET膜为长轴式卷膜，螺旋卷曲后整体形成圆柱状的膜柱，其中，PET膜的结构为规则的纵横交错设置的膜网支架结构；A PET film for cell culture in cell and gene therapy, characterized in that the PET film is a long-axis rolled film, which is spirally rolled to form a cylindrical film column as a whole, wherein the structure of the PET film is a regular criss-cross arranged membrane network support structure;
    该PET膜的膜表面经过硬化处理之后的硬化值在3H-4H之间；The hardening value of the film surface of the PET film after hardening treatment is between 3H and 4H;
    该PET膜内部的纤维呈网格状，且纤维直径在20-30μm范围之间；The fibers inside the PET film are in a grid shape, and the fiber diameter is between 20-30 μm;
    在干燥条件下，该PET膜的拉伸强度在3-6.5MPa范围之间；Under dry conditions, the tensile strength of the PET film is in the range of 3-6.5MPa;
    该PET膜具有亲水性；The PET film is hydrophilic;
    该PET膜整体呈透明无色，透光率大于90%。The PET film is transparent and colorless as a whole, and its light transmittance is greater than 90%.
  2. 根据权利要求1所述的细胞和基因治疗中用于细胞培养的PET膜，其特征在于，所述的PET膜可用于培养的贴壁细胞包括哺乳动物细胞，体细胞，干细胞，祖细胞，成熟细胞，成纤维细胞，骨骼组织，心肌与平滑肌、肝、肺、肾、乳腺皮肤、神经胶质细胞，内分泌细胞，黑色素细胞以及各种肿瘤细胞。The PET membrane for cell culture in cell and gene therapy according to claim 1 is characterized in that the adherent cells that can be cultured on the PET membrane include mammalian cells, somatic cells, stem cells, progenitor cells, mature cells, fibroblasts, bone tissue, myocardial and smooth muscle, liver, lung, kidney, breast skin, glial cells, endocrine cells, melanocytes and various tumor cells.
  3. 根据权利要求2所述的细胞和基因治疗中用于细胞培养的PET膜，其特征在于，成熟细胞可包括神经细胞、心肌细胞、视网膜细胞和肝细胞；The PET film for cell culture in cell and gene therapy according to claim 2, characterized in that the mature cells may include nerve cells, cardiomyocytes, retinal cells and hepatocytes;
    干细胞可包括间充质干细胞、iPS细胞、ES细胞、Muse细胞、胚胎癌症细胞、胚胎生殖干细胞。Stem cells may include mesenchymal stem cells, iPS cells, ES cells, Muse cells, embryonic cancer cells, and embryonic germ stem cells.
  4. 根据权利要求2所述的细胞和基因治疗中用于细胞培养的PET膜，其特征在于，所述的PET膜进行细胞贴附、培养的工作过程为：The PET film for cell culture in cell and gene therapy according to claim 2 is characterized in that the working process of cell attachment and culture of the PET film is:
    将通过各种参数测试后的PET膜置于孔板中，加入适量培养基后，接种细胞，每隔3小时将载体转移至新的孔板，并收集未贴附的细胞进行计数，计算得到贴附率，直至贴附率接近80%；Place the PET membrane that has passed various parameter tests in a well plate, add an appropriate amount of culture medium, and inoculate cells. Transfer the carrier to a new well plate every 3 hours, collect and count the unattached cells, and calculate the attachment rate until the attachment rate is close to 80%;
    继续对上述完成贴附的细胞进行培养，培养过程中定时更换培养基，每隔24小时进行细胞取样，对从PET膜上取下的细胞进行消化裂解后，进行细胞密度的检测，绘制生长曲线。Continue to culture the attached cells, change the culture medium regularly during the culture process, take cell samples every 24 hours, digest and lyse the cells removed from the PET membrane, detect the cell density, and draw a growth curve.
  5. 根据权利要求4所述的细胞和基因治疗中用于细胞培养的PET膜，其特征在于，在PET膜进行细胞贴附之前，对所述PET膜进行碱中和处理、灭菌处理以及支原体检测和细菌内毒素检测；The PET film for cell culture in cell and gene therapy according to claim 4, characterized in that before the PET film is subjected to cell attachment, the PET film is subjected to alkali neutralization treatment, sterilization treatment, mycoplasma detection and bacterial endotoxin detection;
    碱中和处理具体为：0.5M的NaOH溶液完全浸泡PET膜2h后，超纯水循环冲洗2遍，第一次冲洗2h,更换超纯水后再进行二次冲洗4h；The specific alkaline neutralization treatment is as follows: the PET film is completely immersed in a 0.5M NaOH solution for 2 hours, and then rinsed twice with ultrapure water. The first rinse is 2 hours, and then the ultrapure water is replaced and rinsed for a second time for 4 hours.
    灭菌处理具体为：将经过碱中和处理后的PET膜放入高压蒸汽灭菌锅中，压力100kPa，温度121°C，灭菌45min后晾干；The sterilization treatment is specifically as follows: the PET film after alkali neutralization treatment is placed in a high-pressure steam sterilizer at a pressure of 100 kPa and a temperature of 121°C, sterilized for 45 minutes, and then dried;
    支原体检测的标准为无支原体，细菌内毒素检测的标准为内毒素的含量＜EU/ml。The standard for mycoplasma detection is the absence of mycoplasma, and the standard for bacterial endotoxin detection is the endotoxin content <EU/ml.
  6. 根据权利要求4所述的细胞和基因治疗中用于细胞培养的PET膜，其特征在于，所述PET膜进行细胞培养时，对应的容器载体可采用细胞培养皿、细胞培养用方瓶或转瓶、细胞工厂、细胞生物反应器。The PET film for cell culture in cell and gene therapy according to claim 4 is characterized in that when the PET film is used for cell culture, the corresponding container carrier can be a cell culture dish, a square bottle or a rotating bottle for cell culture, a cell factory, or a cell bioreactor.
  7. 根据权利要求4所述的细胞和基因治疗中用于细胞培养的PET膜，其特征在于，所述PET膜进行细胞培养时，可将PET膜裁剪出若干个小型长条取样条，所述长条取样条放置在所述膜柱的内层、中间层以及外层的不同位置。The PET film for cell culture in cell and gene therapy according to claim 4 is characterized in that when the PET film is used for cell culture, the PET film can be cut into a plurality of small long sampling strips, and the long sampling strips are placed at different positions of the inner layer, middle layer and outer layer of the membrane column.
  8. 权利要求1-3中任意一项所述细胞和基因治疗中用于细胞培养的PET膜作为载体，在细胞培养中的应用。Use of the PET film for cell culture in cell and gene therapy as claimed in any one of claims 1 to 3 as a carrier in cell culture.
PCT/CN2023/113930 2022-10-27 2023-08-21 Pet film for cell culture in cell and gene therapy, and use thereof WO2024087828A1 (en)

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