CN116240159A

Movatterモバイル変換

Info

Publication number: CN116240159A
Application number: CN202211492763.3A
Authority: CN
Inventors: 苏杭; 鲜米洗努尔·阿布来提; 李立东
Original assignee: Beijing Taidong Biotechnology Co ltd
Current assignee: Beijing Taidong Biotechnology Co ltd
Priority date: 2022-11-25
Filing date: 2022-11-25
Publication date: 2023-06-09

Abstract

The invention discloses a method for culturing cells on a large scale by using a porous foam metal material, which comprises the following steps: s1, sterilizing and hydrophilic treatment of a porous foam metal material; s2, coating a cell adhesion matrix; s3, inoculating cells, and mixing the cell suspension with the material obtained in the step S1 to obtain a porous foam metal material of the mixed cell suspension; s4, cell culture: step S2, immersing the cells in a complete culture medium after the cells are adhered, and culturing; the whole 3D cell culture scaffold provided by the invention is made of foamed metal aluminum/copper/iron nickel/titanium, and has an average pore diameter of 100-500 mu m and high regularity. The foam metal presents a three-dimensional pore canal structure, has high connectivity, and is convenient for the transmission of nutrient components of each part, the consistency of metabolic activity and the culture result of cells in the 3D culture process; the culture area of the 3D cell culture bracket is far larger than that of a common 2D type cell culture product, so that the space and materials are saved, and the efficiency and the yield of cell culture are greatly improved.

Description

Method for large-scale cell culture by porous foam metal material

Technical Field

The invention relates to the technical field of cell culture, in particular to a method for culturing cells on a large scale by using a porous foam metal material.

Background

In the clinical transformation application process of cell therapy, the culture, passage and expansion processes of cells are an important ring of cell therapy products. The culture modes of the cells are two-dimensional (traditional) culture and three-dimensional culture. Foam metal is composed of a metal skeleton and internal pores, since the advent of the 20 th century, 40, which has a series of excellent properties: low density, high porosity, high specific strength, high specific stiffness, large specific surface area, good energy absorption, damping and vibration reduction performance, corrosion resistance, high temperature resistance, noise reduction and the like, and are widely focused by researchers in various countries, particularly in the fields of preparation technology and performance application research. Currently, most metal foam manufacturing processes are gradually developed and are gradually applied to industrial production. Along with the deep research of various performances, the sock-soaking material is used as a structural material and a functional material and is popularized in the fields of aerospace, electronic communication, construction, transportation, atomic energy, medicine, environmental protection, metallurgy, machinery and the like.

At present, the production process of cell therapy products entering the clinical test stage basically adopts the two-dimensional culture process of a traditional culture bottle, and a small amount of multi-layer cell factories are adopted, namely cells are directly inoculated into a cell culture bottle or a cell culture dish for wall-attached culture, so that the purpose of cell culture expansion is achieved. However, these methods have the disadvantages of small usable cell growth area, large cell culture vessel occupation area, and uneven cell quality during cell harvest.

Meanwhile, the current traditional main cell culture method is 2D culture, which is equivalent to supporting cell growth on only one plane, has low proliferation efficiency and space utilization rate, and cannot meet the requirement of increasing large-dose application of clinical cells.

Disclosure of Invention

The invention aims to provide a method for culturing cells in a large scale by using a porous foam metal material, wherein the porous foam metal material is established, and has extremely strong cell adhesion characteristics without absorbing cytokines and growth factors, so that cells and cell secretion can be directly separated from a 3D bracket during harvesting; can resist high-pressure sterilization, can be reused, and greatly reduces the cost.

In order to achieve the above purpose, the present invention provides the following technical solutions:

a method for culturing cells on a large scale by using a porous foam metal material specifically comprises the following steps:

s1, sterilizing and hydrophilic treatment of a porous foam metal material: sequentially ultrasonically cleaning porous metal by pure water, absolute ethyl alcohol and acetone for 15min, rinsing by deionized water, and standing at high temperature and high pressure for later use; respectively soaking the materials in PBS (phosphate buffer solution) with concentration of 10-100 μg/ml, namely PLL (phase locked loop)/matrigel/fibronectin/vitronectin, performing vibration reaction for 24 hours at 4 ℃, taking out, draining, and soaking in PBS;

s2, cell adherence matrix coating treatment: culturing cells in a cell incubator with 5% CO2 at 37 ℃, observing the growth and proliferation states of the cells under an inverted microscope, changing liquid every other day, and passaging when the cells are adhered to 80% -90%;

s3, inoculating cells, and mixing the cell suspension with the material obtained in the step S1 to obtain a porous foam metal material of the mixed cell suspension;

s4, cell culture: and S2, immersing the cells in a complete culture medium after the cells are adhered, culturing, detecting CCK-8 by using a CCK-8 method when detecting cell proliferation cultures 1, 3, 5 and 7d, detecting absorbance of each group of samples by using an enzyme-labeled instrument, and preparing a cell proliferation curve according to time and absorbance average values.

The density of the cell suspension is 1X 10-4 to 1X 10-8 cells/mL, and the cell suspension is obtained by digestion of adherent cells.

The foam metal material sterilization method comprises the steps of high-pressure steam sterilization, radiation sterilization and disinfectant soaking sterilization. The cell wall-attaching matrix comprises Polylysine (PLL), matrigel (Matrigel), fibronectin (FN), collagen (Collagen), vitronectin (Vitronectin), and Vitronectin (Vitronectin).

The specific implementation method of the step S3 comprises the steps of selecting umbilical mesenchymal stem cells with good growth condition, preparing cell suspension with cell concentration of 1X 10-5/mL, placing the treated porous foam metal material into each hole of a 6-hole plate, sealing the periphery and the bottom by a sterile plastic film, dripping 1mL of wet material of the cell suspension, standing a cell incubator for 2 hours, removing the plastic film, dripping 2mL of culture medium into each hole, continuing to culture at 37 ℃ under the condition of 5% CO2, and simultaneously setting a control group for 2D culture.

Wherein, the cells applicable to the porous foam metal material comprise human embryonic stem cells (hESCs), induced pluripotent stem cells (hiPSCs), retinal pigment epithelial cells (hRPEs), nerve cells (Neuron), mesenchymal stem cells (hMSCs), cardiac muscle cells (cardiomycyte), islet cells, 293T cells (293T) and CHO Cells (CHO).

The porous foam metal material comprises a metal framework and internal holes, wherein the porosity of the porous foam metal material is 50% -98%, and the porosity can be adjusted according to actual requirements.

Wherein the metal framework is a 3D cell culture bracket, the 3D cell culture bracket is made of foam metal, and the average pore diameter is 100-500 mu m, so that the transmission of nutrient components of each part, the consistency of metabolic activity and the culture result of the cells in the 3D culture process are facilitated.

The porous foam metal material takes the foam form of the biomedical metal material as a cell culture framework and comprises biomedical metal foam materials of titanium-based, cobalt-based, magnesium-based, zirconium-based, aluminum alloy and stainless steel structures, and the porous foam metal material has long-term practicability and biocompatibility, and is preferably titanium and titanium alloy.

The porous foam metal material is subjected to high-hydrophilicity cell adherence reagent package treatment, so that the surface of the porous foam metal material can have extremely strong cell adherence characteristic and does not absorb cytokines and growth factors, and cells and cell secretion can be directly separated from the 3D bracket during harvesting.

The porous foam metal material is suitable for culturing central system neurons, collagen (Collagen) is used as an important extracellular matrix protein for supporting cell and tissue growth, and the extracellular environment formed by the Collagen (Collagen) is beneficial to the adhesion, growth, migration and differentiation of various cells.

Compared with the prior art, the invention has the beneficial effects that:

1. the whole 3D cell culture scaffold provided by the invention is made of foamed metal aluminum/copper/iron nickel/titanium, and has an average pore diameter of 100-500 mu m and high regularity. The foam metal presents a three-dimensional pore canal structure, has high connectivity, and is convenient for the transmission of nutrient components of each part, the consistency of metabolic activity and the culture result of cells in the 3D culture process;

2. the culture area of the foammetal cell culture 3D bracket is far larger than that of a common 2D type cell culture product, so that space and materials are saved, and the efficiency and yield of cell culture are greatly improved;

3. unlike conventional 2D monolayer cell culture, 3D stem cell culture techniques refer to the creation of cell spheres by cell aggregation or the embedding of cells on or within a scaffold to mimic ECM (extracellular matrix) of real organism tissue, and thus to some extent the in vivo microenvironment. The 3D culture technology not only has the substance and structure foundation of the in-vivo cell microenvironment, but also can show the intuitiveness of cell culture and the advantage of condition controllability.

Drawings

FIG. 1 is a plan view of a 2D monolayer cell culture scaffold;

FIG. 2 is a plan view of a 3D stem cell culture scaffold employed in the present invention;

FIG. 3 is a graph comparing culture yields of cell culture with a 3D culture scaffold built up of porous metal foam material and a conventional 2D monolayer cell culture scaffold;

FIG. 4 is a graph showing a comparison of the number of harvested cells according to the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the embodiments of the invention

The present invention will be described in further detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Example 1

s1, material treatment: sequentially ultrasonically cleaning porous metal for 15min by pure water, absolute ethyl alcohol and acetone, rinsing by deionized water, and standing at high temperature and high pressure. Soaking the materials in PBS (phosphate buffer solution) with concentration of 10-100 μg/ml, vibration reaction at 4deg.C for 24 hr, taking out, draining, and soaking in PBS;

s2, culturing the cells at 37 ℃ and 5% CO₂ Culturing human embryonic stem cells in a cell incubator, observing the growth and proliferation states of the cells under an inverted microscope, changing liquid every other day, and carrying out passage when the cells are adhered to 80% -90%;

s3, selecting human embryonic stem cells with good growth condition, preparing cell suspension with cell concentration of 1X 10-5/mL, placing the treated porous foam metal material into each hole of a 6-hole plate, sealing the periphery and the bottom by a sterile plastic film, dripping 1mL of soaked material of the cell suspension, standing for 2 hours in a cell incubator, removing the plastic film, dripping 2mL of culture medium into each hole, continuing to culture at 37 ℃ under the condition of 5% CO2, and continuing to culture. Setting a control group of 2D culture at the same time;

and S4, when the CCK-8 method is used for detecting the cell proliferation culture for 1, 3, 5 and 7d, CCK-8 detection is carried out, the absorbance of each group of samples is detected by an enzyme-labeled instrument, and a cell proliferation curve is prepared according to the time and the average value of the absorbance.

Example 2

s2, culturing the cells at 37 ℃ and 5% CO₂ Culturing induced pluripotent stem cells in a cell incubator, observing the growth and proliferation states of the cells under an inverted microscope, changing liquid every other day, and carrying out passage when the cells are adhered to 80% -90%;

s3, selecting induced pluripotent stem cells with good growth condition, preparing cell suspension with cell concentration of 1X 10-5/mL, placing the treated porous foam metal material into each hole of a 6-hole plate, sealing the periphery and the bottom by a sterile plastic film, dripping 1mL of soaked material of the cell suspension, standing for 2h in a cell incubator, removing the plastic film, dripping 2mL of culture medium into each hole, and dripping 5% CO at 37 DEG C₂ Continuing under the condition, and continuing culturing. Setting a control group of 2D culture at the same time;

Example 3

s2, culturing the cells at 37 ℃ and 5% CO₂ Culturing human retinal pigment epithelial cells in a cell incubator, observing the growth and proliferation states of the cells under an inverted microscope, changing liquid every other day, and carrying out passage when the cells are adhered to 80% -90%;

s3, selecting human retinal pigment epithelial cells with good growth condition, preparing cell suspension with cell concentration of 1X 10-5/mL, placing the treated porous foam metal material into each hole of a 6-hole plate, sealing the periphery and the bottom by a sterile plastic film, dripping 1mL of wetted material of the cell suspension, standing for 2h in a cell incubator, removing the plastic film, dripping 2mL of culture medium into each hole, and dripping 5% CO at 37 DEG C₂ Continuing under the condition, and continuing culturing. Setting a control group of 2D culture at the same time;

Example 4

s2, culturing the cells at 37 ℃ and 5% CO₂ Culturing human nerve cells in a cell incubator, observing the growth and proliferation states of the cells under an inverted microscope, changing liquid every other day, and carrying out passage when the cells are adhered to 80% -90%;

s3, selecting human nerve cells with better growth condition, preparing cell suspension with cell concentration of 1X 10-5/ml, and treating the treated porous foam goldPlacing the material into each hole of a 6-hole plate, sealing the periphery and the bottom by using a sterile plastic film, dripping 1mL of wet material of cell suspension, standing a cell incubator for 2 hours, removing the plastic film, dripping 2mL of culture medium into each hole, and dripping 5% CO at 37 DEG C₂ Continuing under the condition, and continuing culturing. Setting a control group of 2D culture at the same time;

Example 5

s2, culturing the cells at 37 ℃ and 5% CO₂ Culturing human mesenchymal stem cells in a cell incubator, observing the growth and proliferation states of the cells under an inverted microscope, changing liquid every other day, and carrying out passage when the cells are adhered to 80% -90%;

s3, selecting human mesenchymal stem cells with better growth condition, preparing cell suspension with cell concentration of 1X 10-5/mL, placing the treated porous foam metal material into each hole of a 6-hole plate, sealing the periphery and the bottom by a sterile plastic film, dripping 1mL of wet material of the cell suspension, standing for 2h in a cell incubator, removing the plastic film, dripping 2mL of culture medium into each hole, and dripping 5% CO at 37 DEG C₂ Continuing under the condition, and continuing culturing. Setting a control group of 2D culture at the same time;

Example 6

s2, culturing the cells at 37 ℃ and 5% CO₂ Culturing human myocardial cells in a cell incubator, observing the growth and proliferation states of the cells under an inverted microscope, changing liquid every other day, and carrying out passage when the cells are adhered to 80% -90%;

s3, selecting human myocardial cells with better growth condition, preparing cell suspension with cell concentration of 1X 10-5/mL, placing the treated porous foam metal material into each hole of a 6-hole plate, sealing the periphery and the bottom by a sterile plastic film, dripping 1mL of soaked material of the cell suspension, standing for 2h in a cell incubator, removing the plastic film, dripping 2mL of culture medium into each hole, and dripping 5% CO at 37 DEG C₂ Continuing under the condition, and continuing culturing. Setting a control group of 2D culture at the same time;

Example 7

s2, culturing the cells at 37 ℃ and 5% CO₂ Culturing human islet cells in a cell incubator, observing the growth and proliferation states of the cells under an inverted microscope, changing liquid every other day, and carrying out passage when the cells are adhered to 80% -90%;

s3, selecting human islet cells with better growth condition, and preparing the islet cells with the cell concentration of 1X 10-5/mlPlacing the treated porous foam metal material into each hole of a 6-hole plate, sealing the periphery and the bottom of the hole by a sterile plastic film, dripping 1mL of soaked material of the cell suspension, standing for 2h in a cell culture box, removing the plastic film, dripping 2mL of culture medium into each hole, and dripping 5% CO at 37 DEG C₂ Continuing under the condition, and continuing culturing. Setting a control group of 2D culture at the same time;

Example 8

s2, culturing the cells at 37 ℃ and 5% CO₂ 293T cells are cultured in a cell incubator, the growth and proliferation states of the cells are observed under an inverted microscope, liquid is changed every other day, and the cells are passaged when 80% -90% of the cells are attached to the wall;

s3, selecting 293T cells with better growth conditions, preparing cell suspension with the cell concentration of 1X 10-5/mL, placing the treated porous foam metal material into each hole of a 6-hole plate, sealing the periphery and the bottom by a sterile plastic film, dripping 1mL of soaked material of the cell suspension, standing for 2 hours in a cell incubator, removing the plastic film, dripping 2mL of culture medium into each hole, and dripping 5% CO at 37 DEG C₂ Continuing under the condition, and continuing culturing. Setting a control group of 2D culture at the same time;

Example 9

1. and (3) material treatment: sequentially ultrasonically cleaning porous metal for 15min by pure water, absolute ethyl alcohol and acetone, rinsing by deionized water, and standing at high temperature and high pressure. Soaking the materials in PBS (phosphate buffer solution) with concentration of 10-100 μg/ml, vibration reaction at 4deg.C for 24 hr, taking out, draining, and soaking in PBS;

2. cell culture at 37℃with 5% CO₂ Culturing CHO cells in a cell incubator, observing the growth and proliferation states of the cells under an inverted microscope, changing liquid every other day, and carrying out passage when the cells are adhered to 80% -90%;

3. selecting CHO cells with good growth condition, preparing cell suspension with cell concentration of 1×10ζ5/mL, placing the treated porous foam metal material into each hole of 6-hole plate, sealing the periphery and bottom with sterile plastic film, dripping 1mL of cell suspension soaked material, standing in cell incubator for 2 hr, removing plastic film, dripping 2mL of culture medium into each hole, 37 deg.C, 5% CO₂ Continuing under the condition, and continuing culturing. Setting a control group of 2D culture at the same time;

and 4, when the CCK-8 method is used for detecting cell proliferation culture for 1, 3, 5 and 7d, CCK-8 detection is carried out, the absorbance of each group of samples is detected by an enzyme-labeled instrument, and a cell proliferation curve is prepared according to the time and the average value of the absorbance.

Specifically, the foam metal material sterilization method comprises the steps of high-pressure steam sterilization, radiation sterilization and disinfectant soaking sterilization. The cell wall-attaching matrix comprises Polylysine (PLL), matrigel (Matrigel), fibronectin (FN), collagen (Collagen), vitronectin (Vitronectin), and Laminin (Laminin).

Specifically, the specific implementation method of the step S3 comprises the steps of selecting umbilical mesenchymal stem cells with good growth condition, preparing cell suspension with cell concentration of 1 x 10-5/mL, placing the treated porous foam metal material into each hole of a 6-hole plate, sealing the periphery and the bottom by a sterile plastic film, dripping 1mL of wet material of the cell suspension, standing a cell incubator for 2 hours, removing the plastic film, dripping 2mL of culture medium into each hole, continuing to culture at 37 ℃ under the condition of 5% CO2, and simultaneously setting a control group for 2D culture.

Specifically, the porous foam metal material comprises a metal framework and internal holes, the porosity of the porous foam metal material is between 50% and 98%, and the porosity can be adjusted according to actual requirements.

Specifically, the metal framework is a 3D cell culture bracket, the 3D cell culture bracket is made of foam metal, and the average pore diameter is 100-500 mu m, so that the transmission of nutrient components of each part, the consistency of metabolic activity and the culture result of the cells in the 3D culture process are facilitated.

Specifically, the porous foam metal material takes the foam form of the biomedical metal material as a cell culture framework, and comprises biomedical metal foam materials of titanium-based, cobalt-based, magnesium-based, zirconium-based, aluminum alloy and stainless steel structures, the porous foam metal material has long-term practicability and biocompatibility, preferably titanium and titanium alloy, and the titanium alloy material is a functional structural material for biomedical engineering and is commonly used for the production and manufacture of surgical implants and orthopedic instrument products. Titanium alloy medical appliance products such as artificial joints, dental implants, vascular stents and the like are used for clinical diagnosis, treatment, repair, replacement of human tissues or organs, or improvement of functions of human tissues or organs, and the functions of the titanium alloy medical appliance products are that medicines cannot be replaced. Pure titanium has the advantages of light weight, no toxicity, light weight, high strength, good biocompatibility and the like. The strength of the pure titanium is 390-490 MPa. Experiments prove that compared with cobalt-based alloy and stainless steel, titanium has better fatigue resistance and corrosion resistance, good surface activity, slight tissue reaction, easy reaction with oxygen to build a compact oxide film, and stable oxide layer of titanium. Therefore, the titanium and the titanium alloy have biomedical material conditions, and are ideal foam metal materials which are suitable for cell culture and have development prospects.

Cobalt-based alloys are commonly referred to as Co-Cr alloys, and have 2 basic designations: co-Cr-Mo alloys and Co-Ni-Cr-Mo alloys. The microstructure of Co-Cr-Mo alloy is cobalt-based austenite structure, can be forged or cast, but is very difficult to manufacture and process, has better mechanical property and corrosion resistance than stainless steel, and is a foam metal material with better current stage.

The stainless steel has low cost, good processability, mechanical property and the like, and is widely applied to the fields of stomatology, fracture internal fixation instruments, artificial joints and the like at present. 302 stainless steel is the earliest medical metal material, and has better corrosion resistance and higher strength. Researchers can add molybdenum element into stainless steel to manufacture 316 stainless steel, so that the corrosion resistance of medical stainless steel can be effectively improved. In the 50 s of the 20 th century, researchers developed a new 316L stainless steel, and the highest carbon content in the stainless steel was reduced to 0.03%, so that the corrosion resistance of the material was further improved. From this point on, medical stainless steel is the material of choice for internationally recognized surgical implants.

The medical stainless steel has the advantages of low price and easy processing, the currently common medical stainless steel is 316L and 317L, the mass fraction of C in the stainless steel is less than or equal to 0.03 percent, the corrosion of the stainless steel in organisms can be avoided, the main components are Fe 60-65 percent, the important alloy Cr 17-20 percent and Ni 12-14 percent are added, and other small element components such as N, mn, mo, P, cl, si and S are added.

Aluminum and its alloy materials have good performance, good plasticity and biocompatibility, and are widely used as implant materials in the early 40 th century, and currently, parts capable of bearing high load are made of aluminum and its alloys. The aluminum has higher corrosion resistance, besides being dissolved in the mixed solution of hydrofluoric acid, caustic alkali, hot concentrated sulfuric acid, hydrochloric acid and nitric acid, other reagents do not have corrosion effect on the aluminum, body fluid does not influence the alternating fatigue strength of the aluminum, and the good biocompatibility ensures that the aluminum foam material does not stimulate cells. In addition, aluminum has a high resistance to notch crack propagation compared to stainless steel.

Specifically, the porous foam metal material is subjected to treatment of a highly hydrophilic cell wall-attaching reagent packet, so that the surface of the porous foam metal material can have extremely strong cell wall-attaching characteristics and does not absorb cytokines and growth factors, and cells and cell secretion can be directly separated from the 3D bracket during harvesting.

In particular, the porous foam metal material is suitable for culturing central system neurons, and uses Collagen (Collagen) as an important extracellular matrix protein for supporting cell and tissue growth, wherein the extracellular environment formed by the Collagen (Collagen) is beneficial to the adhesion, growth, migration and differentiation of various cells.

It should be noted that: the invention takes the foam form of biomedical metal material as cell culture skeleton, which comprises biomedical metal foam materials such as titanium-based, cobalt-based, magnesium-based, zirconium-based, aluminum alloy, stainless steel and the like. The biomedical metal material foam form has the following properties:

(1) The mechanical foam metal material should generally have sufficient strength and toughness, adequate elasticity and hardness, good fatigue and creep resistance, and the necessary wear resistance and self-lubricity.

(2) Corrosion of corrosion-resistant foam metal materials mainly occurs: the surface of the material is exposed in the environment of the cell culture solution to generate electrolysis, which belongs to general uniform corrosion; pitting caused by mixing impurities into the foam material; intergranular corrosion caused by different components and physicochemical properties; the ionization energy is different materials to mix and use the galvanic corrosion that causes; when the foam metal material is loaded, stress corrosion caused by stress concentration occurs at a certain part of the foam metal material; fatigue corrosion and the like which cause damage and fracture of the implant material after long-time repeated loading.

(3) Biocompatibility of

Biocompatibility refers to the degree to which cells and metallic foam material are contained and adapted to each other, i.e., whether the metallic foam material will cause damage, poisoning, and other deleterious effects to the cells. The foam metal material must have excellent biocompatibility, in particular, in that: no toxicity, no irritation, no cancer or mutation to cells; the cells have no rejection reaction; preferably, the binding agent is capable of forming a chemical bond with the cell and is biologically active. Biocompatibility is an important index for measuring the quality of biological materials.

Specifically, cells to which the porous foam metal material is applicable include embryonic stem cells, induced pluripotent stem cells, retinal pigment epithelial cells, nerve cells, mesenchymal stem cells, cardiomyocytes, islet cells, 293T cells, CHO cells.

Poly-L-Lysine (PLL) coating is suitable for most cells, especially for the culture of central system neurons. Collagen (Collagen) is an important extracellular matrix protein that supports cell and tissue growth, and the extracellular environment that it forms facilitates the adhesion, growth, migration and differentiation of a variety of cells. Type I collagen promotes adherence and proliferation of normal and transfected cells. The collagen mainly comprises type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen and type VI collagen. Fibronectin (FN), a macromolecular protein located on the cell surface and in plasma, is a major cell adhesion molecule that plays a structural and adhesive role in the cell fibrous matrix. Vitronectin (Vitonictin) is a small molecule protein that allows cells to attach to the cell matrix via the Vitonictin receptor, and is effective in promoting cell migration, increasing cell adhesion, cell division, cell growth and differentiation, and assisting in the transmission of signals between cells. Laminin (Laminin) is an important component of the extracellular matrix, present on the basal side of all epithelial and endothelial cells, and is also an intermediary for certain cell-cell interactions. Laminin has cell and tissue specificity and plays an important role in regulating cellular functions, such as: assisting adhesion, promoting growth, directing migration, regulating differentiation, maintaining phenotype, preventing apoptosis, etc.

The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Claims

1. A method for large-scale cell culture by using a porous foam metal material, which is characterized by comprising the following steps: the method specifically comprises the following steps:

s2, cell adherence matrix coating treatment: culturing cells in a cell culture box with CO2 concentration of 5% at 37 ℃, observing the growth and proliferation states of the cells under an inverted microscope, changing liquid every other day, and carrying out passage when the cells are adhered to 80% -90%;

s4, cell culture: step S2, after the cells are adhered, immersing the cells in a complete culture medium for culturing, and detecting CCK-8 when detecting cell proliferation culture for 1, 3, 5 and 7d by using a CCK-8 method, detecting absorbance of each group of samples by using an enzyme-labeled instrument, and manufacturing a cell proliferation curve according to time and absorbance average value;

2. The method for large-scale cell culture using a porous metal foam material according to claim 1, wherein: the foam metal material sterilization method comprises the steps of high-pressure steam sterilization, radiation sterilization and disinfectant soaking sterilization. The cell wall-attaching matrix comprises Polylysine (PLL), matrigel (Matrigel), fibronectin (FN), collagen (Collagen), vitronectin (Vitronectin), and Vitronectin (Vitronectin).

3. The method for large-scale cell culture using a porous metal foam material according to claim 1, wherein: the specific implementation method of the step S3 comprises the steps of selecting umbilical mesenchymal stem cells with good growth condition, preparing cell suspension with cell concentration of 1X 10-5/mL, placing the treated porous foam metal material into each hole of a 6-hole plate, sealing the periphery and the bottom by a sterile plastic film, dripping 1mL of wet material of the cell suspension, standing a cell incubator for 2 hours, removing the plastic film, dripping 2mL of culture medium into each hole, continuing to culture at 37 ℃ under the condition of 5% CO2, and simultaneously setting a control group for 2D culture.

4. The method for large-scale cell culture using a porous metal foam material according to claim 1, wherein: cells for which the porous foam metal material is applicable include embryonic stem cells, induced pluripotent stem cells, retinal pigment epithelial cells, neural cells, mesenchymal stem cells, cardiomyocytes, islet cells, 293T cells, CHO cells.

5. The method for large-scale cell culture using a porous metal foam material according to claim 1, wherein: the porous foam metal material comprises a metal framework and internal holes, wherein the porosity of the porous foam metal material is 50% -98%, and the porosity can be adjusted according to actual requirements.

6. The method for large-scale cell culture using a porous metal foam material according to claim 5, wherein: the metal framework is a 3D cell culture bracket, the 3D cell culture bracket is made of foam metal, and the average pore diameter is 100-500 mu m, so that the transmission of nutrient components of each part, the consistency of metabolic activity and the culture result of the cells in the 3D culture process are facilitated.

7. The method for large-scale cell culture using a porous metal foam material according to claim 5, wherein: the porous foam metal material takes the foam form of the biomedical metal material as a cell culture framework and comprises biomedical metal foam materials of titanium-based, cobalt-based, magnesium-based, zirconium-based, aluminum alloy and stainless steel structures, and the porous foam metal material has long-term practicability and biocompatibility, and is preferably titanium and titanium alloy.

8. The method for large-scale cell culture using a porous metal foam material according to claim 1, wherein: the porous foam metal material is subjected to high-hydrophilic cell adherence reagent package treatment, so that the surface of the porous foam metal material can have extremely strong cell adherence characteristic and does not absorb cytokines and growth factors, and cells and cell secretion can be directly separated from the 3D bracket during harvesting.

9. The method for large-scale cell culture using a porous metal foam material according to claim 8, wherein: the porous foam metal material is suitable for cell culture of central system neurons, collagen (Collagen) is used as an important extracellular matrix protein for supporting cell and tissue growth, and the extracellular environment formed by the Collagen (Collagen) is beneficial to adhesion, growth, migration and differentiation of various cells.