Cell culture surface and preparation method thereof
By applying the pre -coating pre -painting and frozen at the grassroots level, it solves the problem of prone transformation, complex operation and uneven surface solution in gel culture technology, and achieves more efficient and stable cells. Cultivation has improved the reliability of the experimental success rate and results.
Patent Information
- Application Number
- CN202411692554.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-25
- Publication Date
- 2025-01-28
AI Technical Summary
The existing gel culture technology has problems such as the volatile gel precursor solution under non-freezing conditions, complex operation, and uneven gel surface, which affects the stability of cell culture and the reliability of experimental results.
The frozen hydrogel anterior body solution is pre -coated and frozen on the grassroots, simplifying the gel coating preparation process, ensuring the stability and biological activity of the gel, and forming a uniform gel surface.
Improve the efficiency and convenience of the experiment, ensure the stability and biological activity of gel, avoid the problem of unevenness on the surface of gel, promote the real reflection of the uniform distribution of cells and the physiological state. sex.
Smart Images
Figure CN119372132A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of cell culture, and specifically relates to a cell culture surface and a preparation method thereof. Background Art
[0002] In biomedical research and clinical applications, the development of cell culture technology is crucial for a deeper understanding of cell behavior and disease mechanisms. Traditional two-dimensional cell culture methods often fail to truly reflect the physiological environment of cells in the body. Gel surface cell culture technology uses protein gels (such as collagen, matrix gel, etc.) to provide cells with a three-dimensional growth environment that is more similar to the internal environment. This technology simulates the natural microenvironment of cells in the body and promotes cell attachment, proliferation and differentiation. Cells can be embedded in gels or grown on their surfaces, and are often used to study tumor biology, stem cell differentiation, and tissue engineering. Gel coatings are widely used in three-dimensional cell culture, in vitro cancer research, intercellular signaling research, low-cost drug screening, biomarker expression, cell invasion ability research, and serum-free cell proliferation.
[0003] However, the existing gel culture technology has the following disadvantages:
[0004] 1. When protein hydrogel precursor solutions (such as matrix gel, etc.) are stored for a long time under non-freezing conditions, they are prone to enzymatic degradation or nonspecific denaturation, resulting in the loss of their structure and function, affecting the stability and biological activity of the gel coating.
[0005] 2. Although some gel precursor solutions can maintain good activity in a non-frozen state, they often need to be pretreated before gelation. For example, thrombin needs to be added to the fibrin precursor solution in advance to help it gel, and collagen gel needs to be neutralized before the precursor solution can gel, which also increases the complexity of the operation.
[0006] 3. The gel precursor solution or the pre-treated gel precursor solution often requires special conditions to activate the gel reaction to guide the solution into a gel solid. Due to the difficulty of controlling the conditions during on-site use, the precursor solution tends to solidify prematurely, which in turn leads to adverse phenomena such as gel wall adhesion and uneven gel surface. Cells will be unevenly distributed on uneven surfaces, and may even show abnormal shapes and sizes, thus affecting the experimental results.
[0007] Therefore, developing cell culture surfaces pre-coated with a frozen gel precursor solution has important practical value. Summary of the invention
[0008] The object of the present invention is to provide a cell culture surface and a preparation method thereof in order to solve the above-mentioned problems.
[0009] The technical scheme adopted by the present invention is as follows: a cell culture surface comprises a base layer and a hydrogel precursor solution in a frozen state.
[0010] The gel is collagen, laminin, entactin, matrix glue, fibrin glue, extracellular matrix extract, polypeptide or a combination thereof
[0011] The base layer is a culture dish, a 384-well plate, a 96-well plate, a 24-well plate, a 6-well plate, a 12-well plate, a 48-well plate, a microcarrier, a pit, a glass bottom plate, a biochip, a cell migration chamber, and a cell culture bottle.
[0012] In a preferred embodiment, the water content of the gel is 10.99-99.99%.
[0013] In a preferred embodiment, the substrate is a culture dish, a 384-well plate, a 96-well plate, a 24-well plate, a 6-well plate, a 12-well plate, a 48-well plate, a microcarrier, a pit, a glass-bottom plate, a biochip, a cell migration chamber, or a cell culture flask.
[0014] In a preferred embodiment, a method for preparing a cell culture surface is characterized in that the method comprises the following steps:
[0015] a) coating the ungelled solution onto a substrate, wherein the substrate is a culture dish, a 384-well plate, a 96-well plate, a 24-well plate, a 6-well plate, a 12-well plate, a 48-well plate, a microcarrier, a pit, a glass-bottomed plate, a biochip, a cell migration chamber, or a cell culture flask;
[0016] b) Before the solution gels, the coated base layer is frozen.
[0017] In a preferred embodiment, a cell culture method comprises:
[0018] a) gelling the cell culture surface according to claim 1;
[0019] b) Place cells on the gel surface for culture.
[0020] In a preferred embodiment, a cell culture surface is used for angiogenesis experiments, cell spheroid experiments, organoid co-culture, cell position fixation, organoid position fixation, cell organoid cryosectioning, stem cell differentiation experiments, tissue engineering applications, and cell invasion experiments.
[0021] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:
[0022] 1. In the present invention, by pre-coating and freezing the hydrogel precursor solution on the base layer, the traditional gel coating preparation process is simplified, avoiding the user from having to perform a cumbersome coating preparation process before use, such as thawing the protein solution or pre-treatment. This ready-to-use design greatly improves the efficiency and convenience of the experiment. At the same time, the pre-coated and frozen hydrogel precursor solution can ensure its stability and biological activity, reduce the reduction of gel function caused by repeated freezing and thawing or improper operation, and thus improve the success rate of the experiment. In addition, the hydrogel provides cells with a three-dimensional growth environment that is more similar to that in the body, promotes cell attachment, proliferation and differentiation, and more realistically reflects the physiological state of cells in the body.
[0023] 2. In the present invention, the pre-coated and frozen hydrogel precursor solution can form a uniform gel surface, avoiding problems such as gel wall hanging or uneven surface, ensuring that cells are evenly distributed during the culture process, which is conducive to obtaining more reliable experimental results. The cell culture surface can be used for a variety of cell culture experiments, including angiogenesis experiments, cell spheroid experiments, organoid co-culture, cell position fixation, organoid position fixation, cell organoid frozen section, stem cell differentiation experiments, tissue engineering applications, cell invasion experiments, etc., and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 It is a schematic diagram of angiogenesis of the present invention;
[0025] Figure 2 This is a schematic diagram of the growth of organoids in the present invention;
[0026] Figure 3 This is a schematic diagram of the staining and counting of cells in the present invention. DETAILED DESCRIPTION
[0027] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0028] Reference Figure 1 ,
[0029] A cell culture surface comprises a base layer and a hydrogel precursor solution in a frozen state.
[0030] The gel is collagen, laminin, entactin, matrix glue, fibrin glue, extracellular matrix extract, polypeptide or a combination thereof
[0031] The base layer is a culture dish, a 384-well plate, a 96-well plate, a 24-well plate, a 6-well plate, a 12-well plate, a 48-well plate, a microcarrier, a pit, a glass bottom plate, a biochip, a cell migration chamber, a cell culture bottle, etc.
[0032] The water content of the gel is 10.99-99.99%.
[0033] The base layer is a culture dish, a 384-well plate, a 96-well plate, a 24-well plate, a 6-well plate, a 12-well plate, a 48-well plate, a microcarrier, a pit, a glass bottom plate, a biochip, a cell migration chamber, and a cell culture bottle.
[0034] The method for preparing the cell culture surface comprises the following steps:
[0035] a) coating the ungelled solution onto a substrate, wherein the substrate is a culture dish, a 384-well plate, a 96-well plate, a 24-well plate, a 6-well plate, a 12-well plate, a 48-well plate, a microcarrier, a pit, a glass-bottomed plate, a biochip, a cell migration chamber, or a cell culture flask;
[0036] b) Before the solution gels, the coated base layer is frozen.
[0037] The culture method comprises:
[0038] a) gelling the cell culture surface according to claim 1;
[0039] b) Place cells on the gel surface for culture.
[0040] The applications include angiogenesis experiments, cell sphere formation experiments, organoid co-culture, cell position fixation, organoid position fixation, cell organoid cryosectioning, stem cell differentiation experiments, tissue engineering applications, and cell invasion experiments.
[0041] Example 1: Preparation of frozen matrix gel-covered 48-well plates:
[0042] Liquid matrix gel was added to a pre-cooled 48-well plate, 100 μL per well, and then immediately transferred to a -20°C refrigerator for freezing to obtain a frozen matrix gel-covered 48-well plate.
[0043] Example 2: Preparation of frozen matrix gel-covered cell migration chamber:
[0044] Prepare a 1:15 diluted matrix gel water solution, add the diluted matrix gel into the pre-cooled cell migration chamber, 100 μL per chamber, and then immediately transfer to a -20°C refrigerator for freezing to obtain a frozen matrix gel-covered cell migration chamber.
[0045] Example 3: Treatment of frozen matrix gel-covered cell culture surfaces and their use in an in vitro human umbilical vein endothelial cell (HUVEC) angiogenesis assay:
[0046] The frozen matrix gel-covered 48-well plate in Example 1 was taken out from the low temperature environment and immediately transferred to a 37° C. constant temperature cell culture incubator for incubation for 60 minutes.
[0047] 40,000 HUVEC cells were added to 400 μL of complete culture medium for human macrovascular endothelial cells and mixed well. After the matrix gel in the 48-well plate was formed, the cell suspension was added. The plate was placed in a 37°C constant temperature cell culture incubator for overnight incubation. The angiogenesis was observed using an inverted microscope the next day.
[0048] Example 4: Frozen Matrigel-covered cell culture surface for fixation of organoid positions (using mouse small intestinal organoids as an example):
[0049] The frozen matrix gel-covered 48-well plate in Example 1 was taken out from the low temperature environment and immediately transferred to a 37° C. constant temperature cell culture incubator for incubation for 60 minutes.
[0050] After the matrix gel in the 48-well plate is formed, the mouse small intestinal organoids are inoculated onto the surface of the gel coating, and 400 μL of small intestinal organoid complete culture medium is added. Place in a 37°C constant temperature cell culture incubator and incubate for 3-7 days. The culture medium is replaced every other day, and the growth of the organoids is observed using an inverted microscope.
[0051] Example 5: Cell invasion (taking MDA-MB-231 cells as an example)
[0052] The frozen matrix gel-covered cell migration chamber in Example 2 was taken out from the low temperature environment and immediately transferred to a 37° C. constant temperature cell culture incubator. After incubation for 60 minutes, the excess liquid was discarded.
[0053] Add 30,000 MDA-MB-231 cells to 500 μL of DMEM basal medium (without serum), mix well, and transfer to the chamber. Add 500 μL of DMEM complete medium (with serum) to a 24-well plate, and place the chamber in the 24-well plate. Place in a 37°C constant temperature cell culture incubator and incubate for 30 hours.
[0054] Wipe the gel and cells inside the chamber clean with a clean cotton swab, and use crystal violet to stain and count the cells that have passed through the chamber membrane (see Figure 3 ).
[0055] In the present invention, by pre-coating and freezing the hydrogel precursor solution on the base layer, the traditional gel coating preparation process is simplified, avoiding the user from having to perform a cumbersome coating preparation process before use, such as thawing the protein solution or pre-treatment. This ready-to-use design greatly improves the efficiency and convenience of the experiment. At the same time, the pre-coated and frozen hydrogel precursor solution can ensure its stability and biological activity, reduce the reduction of gel function caused by repeated freezing and thawing or improper operation, and thus improve the success rate of the experiment. In addition, the hydrogel provides cells with a three-dimensional growth environment that is more similar to that in vivo, promotes cell attachment, proliferation and differentiation, and more realistically reflects the physiological state of cells in vivo.
[0056] In the present invention, the pre-coated and frozen hydrogel precursor solution can form a uniform gel surface, avoiding problems such as gel wall hanging or surface unevenness, ensuring that cells are evenly distributed during the culture process, and facilitating obtaining more reliable experimental results. The cell culture surface can be used for a variety of cell culture experiments, including angiogenesis experiments, cell spheroid experiments, organoid co-culture, cell position fixation, organoid position fixation, cell organoid frozen sections, stem cell differentiation experiments, tissue engineering applications, cell invasion experiments, etc., and has broad application prospects.
[0057] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the term "comprise" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.
[0058] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A cell culture surface, characterized in that: A hydrogel precursor solution including a base layer and a frozen state; The gel is collagen, laminin, entactin, matrix glue, fibrin glue, extracellular matrix extract, polypeptide or a combination thereof The base layer is a culture dish, a 384-well plate, a 96-well plate, a 24-well plate, a 6-well plate, a 12-well plate, a 48-well plate, a microcarrier, a pit, a glass bottom plate, a biochip, a cell migration chamber, and a cell culture bottle.
2. The method for preparing a cell culture surface according to claim 1, characterized in that: The water content of the gel is 10.99-99.99%.
3. A cell culture surface and a method for preparing the same as claimed in claim 1, characterized in that: The base layer is a culture dish, a 384-well plate, a 96-well plate, a 24-well plate, a 6-well plate, a 12-well plate, a 48-well plate, a microcarrier, a pit, a glass bottom plate, a biochip, a cell migration chamber, and a cell culture bottle.
4. The method for preparing a cell culture surface according to claim 1, characterized in that: The method comprises the following steps: a) coating the ungelled solution onto a substrate, wherein the substrate is a culture dish, a 384-well plate, a 96-well plate, a 24-well plate, a 6-well plate, a 12-well plate, a 48-well plate, a microcarrier, a pit, a glass-bottomed plate, a biochip, a cell migration chamber, or a cell culture flask; b) Before the solution gels, the coated base layer is frozen.
5. A cell culture method, characterized in that: The culture method comprises: a) gelling the cell culture surface according to claim 1; b) Place cells on the gel surface for culture.
6. An application of a cell culture surface, characterized in that: The applications include using the cell culture surface as described in claim 1 to perform angiogenesis experiments, cell spheroid experiments, organoid co-culture, cell position fixation, organoid position fixation, cell organoid cryosectioning, stem cell differentiation experiments, tissue engineering applications, and cell invasion experiments.