A culture medium, oral mucosal epithelial cell model, and preparation method and use thereof

Abstract

The present invention provides a culture medium, an oral mucosal epithelial cell model, and methods for preparing and using the same, belonging to the field of biotechnology. The present invention successfully constructs a culture medium, using which a three-dimensional in vitro oral mucosal epithelial model with a structure and function highly similar to human oral mucosal epithelial tissue is constructed. This culture medium can be widely promoted and used worldwide, with readily available experimental materials, low cost, and a short cycle time. It can meet various research needs related to oral mucosal epithelial function, is of great significance for advancing oral medicine research and clinical treatment, and has good application prospects in the preparation of drug screening and anti-tumor drug models.

Description

Technical Field

[0001] The present invention belongs to the field of material processing, and specifically relates to a culture medium, an oral mucosal epithelial cell model, and a preparation method and use thereof. Background Art

[0002] The oral mucosal epithelium serves as the body's first line of defense, and its structural and functional integrity is crucial for maintaining oral health. Research on the regulation of oral mucosal epithelial function typically uses clinical biopsy tissue or selected animal model tissues. These methods present drawbacks such as limited operability, significant inter-individual variability, and ethical risks. Therefore, a widely applicable research model and strategy is urgently needed. The construction of a three-dimensional in vitro oral mucosal epithelial model can support the study of mucosal epithelial function, the exploration of disease mechanisms, and the development of new therapies. Currently, common oral mucosal epithelial research models include two-dimensional cell culture and three-dimensional cell-based culture. The cell biological characteristics and cell behavior of two-dimensional models differ significantly from those in vivo, and they are unable to simulate the complex physiological and pathological microenvironment. While existing three-dimensional models have improved in simulating the tissue microenvironment, their construction methods are complex, costly, and limited by experimental techniques and materials, hindering their widespread use.

[0003] Traditional human oral keratinocyte (HOK) culture medium is typically based on a serum-free KSFM medium supplemented with 1% penicillin / streptomycin (P / S). This basic culture medium has the following problems when constructing 3D models: (1) the cell state is unstable, and cells may fall off due to vibration, resulting in poor cell adhesion; (2) it cannot fully simulate the physiological environment of HOK cells in vivo, especially when studying cell differentiation and 3D tissue formation, and cannot form a good 3D structure. Summary of the Invention

[0004] To address the challenges of the prior art, the present invention aims to construct a highly reproducible, easily manipulated, and universally applicable in vitro three-dimensional model using the most widely used oral keratinocyte cell line. This provides technical support for the study of oral epithelial function and therapeutic options. The present invention provides a culture medium, an oral epithelial cell model, and methods for preparing and using the same.

[0005] The present invention provides a culture medium, which consists of a basal culture medium, non-essential amino acids, a glucocorticoid, a cell metabolism supplement, a nucleic acid base supplement, a calcium ion supplement, vitamin C and serum; wherein the concentration of the non-essential amino acids is 2-6 mM, the concentration of the glucocorticoid is 1-2 mM, the concentration of the cell metabolism supplement is 0.05-0.15 mM, the concentration of the nucleic acid base supplement is 0.1-0.3 mM, the calcium ion concentration in the calcium ion supplement is 0-2 mM, the concentration of the vitamin C is 0-50 ug / ml, and the volume of the serum is 0-2% of the total volume of the culture medium.

[0006] Furthermore, the concentration of the non-essential amino acids is 4 mM, the concentration of the glucocorticoid is 1.48 mM, the concentration of the cell metabolism supplement is 0.1 mM, the concentration of the nucleic acid base supplement is 0.18 mM, the calcium ion concentration in the calcium ion supplement is 0~1mM, the concentration of the vitamin C is 0~50 ug / ml, and the volume of the serum is 0~2% of the total volume of the culture medium.

[0007] Furthermore,

[0008] The basal culture medium is a DMEM / F12 (1:1) culture medium in which DMEM culture medium and F12 culture medium are mixed in a ratio of 1:1;

[0009] The non-essential amino acid is L-glutamine;

[0010] The glucocorticoid is hydrocortisone;

[0011] The cell metabolism supplement is O-phosphoethanolamine;

[0012] The nucleic acid base supplement is adenine;

[0013] The calcium ion supplement is calcium chloride;

[0014] The serum is fetal bovine serum.

[0015] Furthermore, the concentration of the non-essential amino acids is 4 mM, the concentration of the glucocorticoid is 1.48 mM, the concentration of the cell metabolism supplement is 0.1 mM, the concentration of the nucleic acid base supplement is 0.18 mM, the calcium ion concentration in the calcium ion supplement is 0.25 mM, the concentration of the vitamin C is 50 ug / ml, and the volume of the serum is 0% of the total volume of the culture medium;

[0016] Alternatively, the concentration of the non-essential amino acids is 4 mM, the concentration of the glucocorticoid is 1.48 mM, the concentration of the cell metabolism supplement is 0.1 mM, the concentration of the nucleic acid base supplement is 0.18 mM, the calcium ion concentration in the calcium ion supplement is 1.0 mM, the concentration of the vitamin C is 50 ug / ml, and the volume of the serum is 0% of the total volume of the culture medium;

[0017] Alternatively, the concentration of the non-essential amino acids is 4 mM, the concentration of the glucocorticoid is 1.48 mM, the concentration of the cell metabolism supplement is 0.1 mM, the concentration of the nucleic acid base supplement is 0.18 mM, the calcium ion concentration in the calcium ion supplement is 0 mM, the concentration of the vitamin C is 50 ug / ml, and the volume of the serum is 1% of the total volume of the culture medium;

[0018] Alternatively, the concentration of the non-essential amino acids is 4 mM, the concentration of the glucocorticoid is 1.48 mM, the concentration of the cell metabolism supplement is 0.1 mM, the concentration of the nucleic acid base supplement is 0.18 mM, the calcium ion concentration in the calcium ion supplement is 0.25 mM, the concentration of the vitamin C is 50 ug / ml, and the volume of the serum is 1% of the total volume of the culture medium;

[0019] Alternatively, the concentration of the non-essential amino acid is 4 mM, the concentration of the glucocorticoid is 1.48 mM, the concentration of the cell metabolism supplement is 0.1 mM, the concentration of the nucleic acid base supplement is 0.18 mM, the calcium ion concentration in the calcium ion supplement is 1.0 mM, the concentration of the vitamin C is 50 ug / ml, and the volume of the serum is 1% of the total volume of the culture medium.

[0020] The present invention also provides a method for preparing the culture medium, which comprises the following steps: weighing raw materials of the culture medium in proportion, and mixing them to obtain the culture medium.

[0021] The present invention also provides use of the culture medium in preparing an oral mucosal epithelial cell model.

[0022] The present invention also provides an oral mucosal epithelial cell model, which is a model obtained by culturing oral mucosal epithelial cells using the above culture medium.

[0023] Furthermore, the oral mucosal epithelial cells are human oral keratinocytes HOK, human immortalized keratinocytes Hacat, human normal squamous epithelial cells NOK, abnormally proliferating oral mucosal keratinocytes DOK or tumor cells Cal27.

[0024] The present invention also provides a method for constructing the above-mentioned oral mucosal epithelial cell model, which comprises the following steps: using an air-liquid interface culture method, on day 0, cells are inoculated into a culture chamber, and the above-mentioned culture medium is added to the upper and lower layers; on day 2, an air interface is constructed and the upper layer of culture medium is removed; on days 12 to 16, the lower layer of culture medium is replaced for culture; after the culture is completed, the three-dimensional tissue is collected and processed to obtain an oral mucosal epithelial cell model.

[0025] The present invention also provides the use of the oral mucosal epithelial cell model in drug screening and anti-tumor drug efficacy.

[0026] The present invention has achieved the following beneficial effects:

[0027] The present invention provides a culture medium, which is used to successfully construct an in vitro three-dimensional oral mucosal epithelium model that is highly similar in structure and function to human oral mucosal epithelial tissue. The culture medium can be widely promoted and used worldwide. The experimental materials are easy to obtain, the cost is low, and the cycle is short. It can meet various research needs related to the function of oral mucosal epithelium, is of great significance for promoting the progress of oral medicine research and clinical treatment, and has good application prospects in the preparation of drug screening and anti-tumor drug models.

[0028] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0029] The following is a further detailed description of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram for the construction of a 3D cell model.

[0031] Figure 2 To investigate the effects of different culture medium compositions on the construction of three-dimensional culture models of HOK cells.

[0032] Figure 3 To investigate the effects of different culture medium compositions on the construction of three-dimensional culture models of Hacat cells.

[0033] Figure 4 To investigate the effects of different culture medium compositions on the construction of three-dimensional culture models of NOK cells.

[0034] Figure 5 To investigate the effects of different culture medium compositions on the construction of three-dimensional culture models of DOK cells.

[0035] Figure 6 To investigate the effects of different culture medium compositions on the three-dimensional culture model of tumor cells Cal27.

[0036] Figure 7 A 3D model constructed for tumor cells Cal27 was used to verify the efficacy of anti-tumor drugs: (A) Representative 3D model staining; (B) Thickness statistics of the 3D model after treatment with different drugs. DETAILED DESCRIPTION

[0037] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0038] The present invention purchases the most commonly used cell lines in oral medicine research, including: human oral keratinocytes HOK, human immortalized keratinocytes Hacat, human normal squamous epithelial cells NOK, abnormally proliferating oral mucosal keratinocytes DOK and tumor cells Cal27.

[0039] DMEM / F12 (1:1) medium refers to a mixture of DMEM medium and F12 medium in a ratio of 1:1, F12 (Sigma-Aldrich, N6658, 500 ml), DMEM (Gbico, C11995500BT, 500 ml).

[0040] Example 1. Preparation of cell culture medium

[0041] 500 ml of DMEM / F12 (1:1) medium was taken as the basal medium, and L-glutamine at a final concentration of 4 mM, hydrocortisone at a final concentration of 1.48 μM, O-phosphoethanolamine at a final concentration of 0.1 mM, and adenine at a final concentration of 0.18 mM were added thereto. The calcium chloride, L-ascorbic acid, and serum contents were adjusted as shown in Table 1 to obtain the cell culture medium of the present invention.

[0042] Table 1 Formulations of different cell culture media

[0043]

[0044] Example 2: Establishment of an in vitro three-dimensional HOK cell culture model

[0045] Cell culture conditions: 37°C, 5% CO2.

[0046] according to Figure 1The cell model was constructed as shown in the schematic diagram. The specific steps were as follows: Using a 24-well cell culture chamber, HOK cells were plated on Day 0 and Medium 1 was added to the upper and lower layers, maintaining a liquid interface between the upper and lower layers. On Day 2, an air interface was established and the upper layer of medium was removed from the chamber. The lower layer of medium was replaced daily from Day 12 to Day 16. Finally, the multilayered 3D tissue cultured in the chamber was collected, fixed, embedded, and sectioned to create a 3D HOK cell culture model.

[0047] Example 3: Establishment of an in vitro three-dimensional culture model of Hacat cells

[0048] Referring to the method of Example 2, an in vitro three-dimensional culture model of Hacat cells was established, except that medium 1 was replaced by medium 2, and HOK cells were replaced by Hacat cells, to obtain a three-dimensional culture model of Hacat cells.

[0049] Example 4: Establishment of an in vitro three-dimensional NOK cell culture model

[0050] Referring to the method of Example 2, an in vitro three-dimensional culture model of Hacat cells was established, except that medium 1 was replaced by medium 3 and HOK cells were replaced by NOK cells, thereby obtaining a three-dimensional culture model of NOK cells.

[0051] Example 5: Establishment of an in vitro three-dimensional DOK cell culture model

[0052] Referring to the method of Example 2, an in vitro three-dimensional culture model of DOK cells was established, except that medium 1 was replaced by medium 4, and HOK cells were replaced by DOK cells, to obtain a three-dimensional culture model of DOK cells.

[0053] Example 6: Establishment of an in vitro three-dimensional culture model of Cal27 cells

[0054] With reference to the method of Example 2, an in vitro three-dimensional culture model of Hacat cells was established, except that medium 1 was replaced by medium 5, and HOK cells were replaced by Cal27 cells, thereby obtaining a three-dimensional culture model of Cal27 cells.

[0055] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0056] Experimental Example 1: Screening of Conditions for Constructing a Three-Dimensional Cell Culture Model in Vitro

[0057] 1. Experimental methods

[0058] 1.1 Hematoxylin-eosin staining

[0059] (1) Place the paraffin sections in a 65°C constant temperature oven and bake for 1.5 h;

[0060] (2) Paraffin sections were dewaxed and hydrated in sequence (dewaxing in xylene I solution for 20 min, dewaxing in xylene II solution for 15 min, and hydrating in 100% I, 100% II, 95%, 80%, and 75% graded alcohol for 5 min each);

[0061] (3) Rinse with distilled water 3 times, 5 min each time;

[0062] (4) Mayer hematoxylin staining for 1 min, 1% hydrochloric acid alcohol staining for 3-5 s, and tap water anti-blueing for 15 min;

[0063] (5) Counterstain with 0.5% eosin for 1–2 min;

[0064] (6) Dehydration in 80%, 90%, and 100% alcohol for 5 min each;

[0065] (7) Transparent in xylene I or II solution, and seal with neutral gum;

[0066] (8) Observe under a microscope.

[0067] 1.2 Immunofluorescence staining

[0068] Cells were cultured on coverslips placed in 24-well plates. Plasmid transfection was then performed using Lipofectamine 2000 (Invitrogen). Eight hours after transfection, cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with PBS containing 0.25% Triton X-100. Next, cells were blocked with 10% goat serum and incubated with a primary antibody against the HA tag overnight at 4°C. Cells were then washed with PBS and incubated with the ALEXAFLUOR 488 secondary antibody. Finally, slides were mounted using ProLong® Gold Antifade reagent containing DAPI (Invitrogen), and images were captured using confocal imaging on an S3000 confocal microscope (HOOKE Instruments).

[0069] 2. Experimental results

[0070] (1) Screening of HOK cell culture conditions

[0071] Refer to the method in Example 1, except that the HOK cell three-dimensional culture model was established according to the culture medium composition shown in Table 2.

[0072] Table 2. Effects of different culture medium compositions on the HOK cell 3D culture model

[0073]

[0074] Note: “-” means the substance is not added.

[0075] Through Table 2 and Figure 2 The three-dimensional model of HOK cells was constructed with different culture medium compositions, and it can be seen that: Ca 2+ Ion concentration will affect the layered structure of the three-dimensional model. 2+ At a concentration of 0.25mM and a vitamin C concentration of 50ug / ml in the absence of serum, the oral mucosal epithelial model constructed with HOK cells exhibited significant stratification, resulting in a more robust three-dimensional model. Compared to three-dimensional models constructed using traditional HOK culture medium, the model demonstrated superior stratification and a more realistic representation of the state of the oral mucosal tissue.

[0076] (2) Screening of Hacat cell culture conditions

[0077] Refer to the method in Example 1, except that the three-dimensional culture model of Hacat cells was established according to the culture medium composition shown in Table 3.

[0078] Table 3. Effects of different culture medium compositions on the three-dimensional culture model of Hacat cells

[0079]

[0080] Note: “-” means the substance is not added.

[0081] Through Table 3 and Figure 3 The three-dimensional model of Hacat cells was constructed with different culture medium compositions, and it can be seen that: Ca 2+ Ion and serum concentrations affect the layered structure of the three-dimensional model. 2+ When the concentration was 1mM and the Vc concentration was 50ug / ml, and there was no serum, the oral mucosal epithelial model constructed by Hacat cells showed significant stratification characteristics and could obtain a better three-dimensional structural model.

[0082] (3) Screening of NOK cell culture conditions

[0083] The method in Reference Example 1 was used, except that the three-dimensional culture model of NOK cells was established according to the culture medium composition shown in Table 4.

[0084] Table 4. Effects of different culture medium compositions on the NOK cell 3D culture model

[0085]

[0086] Note: “-” means the substance is not added.

[0087] Through Table 4 and Figure 4 The three-dimensional model of NOK cells was constructed with different culture medium compositions. It can be seen that serum concentration affects the hierarchical structure of the three-dimensional model. 2+ When the concentration was 0mM, the Vc concentration was 50ug / ml, and the serum was 1%, the oral mucosal epithelial model constructed by NOK cells showed significant stratification characteristics and could obtain a better three-dimensional structural model.

[0088] (4) Screening of DOK cell culture conditions

[0089] Refer to the method in Example 1, except that the DOK cell three-dimensional culture model was established according to the culture medium composition shown in Table 5.

[0090] Table 5. Effects of different culture medium compositions on the DOK cell 3D culture model

[0091]

[0092] Through Table 5 and Figure 5 The three-dimensional model of DOK cells was constructed with different culture medium compositions. It can be seen that serum concentration affects the hierarchical structure of the three-dimensional model. 2+ When the concentration was 0.25mM, the Vc concentration was 50ug / ml, and the content of 1% serum was present, the oral mucosal epithelial model constructed by DOK cells showed significant stratification characteristics and could obtain a better three-dimensional structural model.

[0093] (5) Screening of Cal27 cell culture conditions

[0094] The method in Reference Example 1 was used, except that the Cal27 cell three-dimensional culture model was established according to the culture medium composition shown in Table 6.

[0095] Table 6. Effects of different culture medium compositions on the Cal27 cell 3D culture model

[0096]

[0097] Note: “-” means the substance is not added.

[0098] Through Table 6 and Figure 6 The three-dimensional model of Cal27 cells was constructed with different culture medium compositions. It can be seen that serum concentration affects the hierarchical structure of the three-dimensional model. 2+ When the concentration was 1 mM, the Vc concentration was 50 ug / ml, and the serum contained 1%, the oral mucosal epithelial model constructed by Cal27 cells showed significant stratification characteristics and could obtain a better three-dimensional structural model.

[0099] Experimental Example 2: Application of the in vitro Cal27 cell three-dimensional culture model in the study of anti-tumor drug efficacy

[0100] Based on the three-dimensional culture model constructed for Cal27 cells, the present invention studied the combined effects of two anti-tumor drugs. The advantage of this model is that the culture time is significantly longer than that of the two-dimensional cell level, so long-term drug treatment studies can be performed in vitro. The present invention evaluated the proliferation of cells under the conditions of drug 1 (Drug1), drug 2 (Drug2), and combination therapy (Comb), with CON as the control study group. Over a 14-day period, the results showed that the combination of the two drugs had a better ability to inhibit Cal27 cell proliferation ( Figure 7 A) By statistically analyzing the thickness of the three-dimensional model under different conditions, it was found that the combined use of drugs significantly reduced the three-dimensional structure formed by Cal27 ( Figure 7 B). The above studies show that this model can be widely used in drug screening, anti-tumor drug efficacy research, etc.

[0101] In summary, the present invention found that HOK cells were screened using the culture medium formula (Ca 2+ The concentration was 0.25 mM, the Vc concentration was 50 ug / ml, and serum was not present). Hacat cells were cultured using the selected culture medium formula (Ca 2+ The NOK cells were cultured using a culture medium formulated with a concentration of 1.0 mM and a Vc concentration of 50 μg / ml in the absence of serum. 2+ The DOK cells were cultured using the selected culture medium formula (Ca 2+ The Cal27 cells were cultured in a culture medium with a concentration of 0.25 mM, a Vc concentration of 50 ug / ml, and 1% serum. 2+ At a concentration of 1.0 mM, a Vc concentration of 50 ug / ml, and 1% serum), a significant stratification characteristic can be exhibited in the oral mucosal epithelial model, resulting in a better three-dimensional structural model.

[0102] In summary, the present invention provides a culture medium, an oral mucosal epithelial cell model, and its preparation method and use. The present invention successfully constructed a culture medium, using which an in vitro three-dimensional oral mucosal epithelial model with a structure and function highly similar to human oral mucosal epithelial tissue was successfully constructed. This culture medium can be widely promoted and used worldwide. The experimental materials are easily accessible, the cost is low, and the cycle is short. It can meet the needs of various research related to oral mucosal epithelial function, is of great significance for promoting the progress of oral medicine research and clinical treatment, and has good application prospects in the preparation of drug screening and anti-tumor drug models.

Claims

1. A culture medium for oral mucosal epithelial cells, characterized in that The invention comprises a basal culture medium, L-glutamine, hydrocortisone, O-phosphoethanolamine, adenine, calcium chloride, vitamin C and fetal bovine serum; wherein the basal culture medium is a DMEM / F12 (1:1) culture medium obtained by mixing DMEM culture medium and F12 culture medium in a ratio of 1:1; When the oral mucosal epithelial cells are human oral keratinocytes (HOK), the concentration of L-glutamine is 4 mM, the concentration of hydrocortisone is 1.48 mM, the concentration of O-phosphoethanolamine is 0.1 mM, the concentration of adenine is 0.18 mM, the concentration of calcium ions in calcium chloride is 0.25 mM, the concentration of vitamin C is 50 ug / ml, and the volume of fetal bovine serum is 0% of the total volume of the culture medium; Alternatively, when the oral mucosal epithelial cells are normal human squamous epithelial NOK cells, the concentration of L-glutamine is 4 mM, the concentration of hydrocortisone is 1.48 mM, the concentration of O-phosphoethanolamine is 0.1 mM, the concentration of adenine is 0.18 mM, the calcium ion concentration in calcium chloride is 0 mM, the concentration of vitamin C is 50 ug / ml, and the volume of fetal bovine serum is 1% of the total volume of the culture medium; Alternatively, when the oral mucosal epithelial cells are DOK cells, the concentration of L-glutamine is 4 mM, the concentration of hydrocortisone is 1.48 mM, the concentration of O-phosphoethanolamine is 0.1 mM, the concentration of adenine is 0.18 mM, the calcium ion concentration in the calcium chloride is 0.25 mM, the concentration of vitamin C is 50 ug / ml, and the volume of fetal bovine serum is 1% of the total volume of the culture medium.

2. A method for preparing a culture medium for oral mucosal epithelial cells according to claim 1, characterized in that: The method comprises the following steps: weighing raw materials of the culture medium in proportion, and mixing them to obtain the product.

3. Use of the culture medium of oral mucosal epithelial cells according to claim 1 in preparing an oral mucosal epithelial cell model.

4. An oral mucosal epithelial cell model, characterized in that: The model is obtained by culturing oral mucosal epithelial cells using the culture medium for oral mucosal epithelial cells described in claim 1.

5. The oral mucosal epithelial cell model according to claim 4, characterized in that The oral mucosal epithelial cells are human oral keratinocytes HOK, human normal squamous epithelial cells NOK and DOK cells.

6. A method for constructing the oral mucosal epithelial cell model according to claim 4 or 5, characterized in that: The method comprises the following steps: using an air-liquid interface culture method, on day 0, inoculating cells into a culture chamber, and adding the culture medium of the oral mucosal epithelial cells according to claim 1 to the upper and lower layers; on day 2, establishing an air interface and removing the upper layer of culture medium; replacing the lower layer of culture medium for culturing from day 12 to day 16; after the completion of the culture, collecting and processing the three-dimensional tissue to obtain an oral mucosal epithelial cell model.

7. Use of the oral mucosal epithelial cell model according to claim 4 or 5 in drug screening.

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