CN107164309B - 3D model constructed outside serum-containing culture liquid and construction method thereof - Google Patents

3D model constructed outside serum-containing culture liquid and construction method thereof Download PDF

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CN107164309B
CN107164309B CN201710460520.4A CN201710460520A CN107164309B CN 107164309 B CN107164309 B CN 107164309B CN 201710460520 A CN201710460520 A CN 201710460520A CN 107164309 B CN107164309 B CN 107164309B
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何欣
李潇
卢永波
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Guangdong Biocell Biotechnology Co ltd
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Abstract

The invention discloses a 3D model constructed in vitro by utilizing a serum-containing culture system and a construction method thereof, which relate to the technical field of tissue engineering biological materials, and the 3D model has various seed cell selections and good in vitro construction repeatability, so that industrialized preparation can be realized; meanwhile, the serum-containing culture system is used for providing nutrition required by cells, and simultaneously reducing the addition of factors. The model uses the principle of cell biology and engineering, uses the primary cornea epithelial cells, or limbal stem cells, or immortalized cornea epithelial cells, or primary oral mucosa epithelial cells, or oral mucosa squamous cancer cell line TR146, and adopts serum-containing culture solution and submerged-gas liquid level sectional culture method to make it be stratified after in vitro amplification, so as to form the in vitro 3D model.

Description

3D model constructed outside serum-containing culture liquid and construction method thereof
Technical Field
The invention belongs to the technical field of biological materials in tissue engineering, and particularly relates to a 3D model constructed in vitro by using a serum-containing culture system and a construction method thereof.
Background
The 3D model is constructed by amplifying a small amount of seed cells in vitro by applying the principles of cell biology and engineering, has a structure which is highly similar to that of the cornea epithelium of a normal person, and sequentially comprises a basal layer, a ratchet layer, a granular layer and a ratchet layer from bottom to top, has no stratum corneum, has a complete epithelium structure and expresses cornea related keratins. The 3D model constructed in vitro is mainly used for in vitro detection of eye irritation and is used for evaluating the safety of products such as small molecular compounds, active proteins, medical instruments, chemicals, cosmetics and the like.
Direct contact or exposure of chemicals to the eye can cause irritation and local toxicity. Therefore, ocular surface irritation risk assessment is a highly desirable item in hazard assessment and risk avoidance programs. The evaluation of the eye irritation potential risk of the novel compounds in the traditional method mainly relies on in vitro rabbit eye experiments, which is time-consuming, labor-consuming and costly. Therefore, searching for a rapid and economical compound screening method is an urgent need in the field, and has motivated the development of a great deal of related works about the establishment of in vitro alternative methods for evaluating ocular surface irritation risk, such as bovine cornea opacity and permeability detection, chick embryo allantoic membrane experiments, etc., which can realize in vitro ocular irritation evaluation by a certain degree of combination, but cannot completely realize the replacement of in vitro rabbit ocular irritation experiments. In recent years, with the development of in vitro organ culture technology, a 3D recombinant cornea epithelial model constructed based on in vitro cell culture shows a characteristic trend in the field of evaluation of eye irritation risk. The 3D cornea epithelial model constructed in vitro is similar to the three-dimensional structure of human cornea epithelium, can reflect the response of human body to chemicals more truly, accurately gives out the prediction of chemical irritation, has wider application range for the type of compound screening, and comprises the hazard evaluation of specific components of cosmetics besides the formulation development of different efficacy purposes. Currently, eye irritation assays using in vitro construction of 3D recombinant cornea models have been internationally accepted, and an alternative method for using such models is given by OECD guideline 492 in 2015.
Currently, there are mainly three commercially available in vitro recombinant corneal epithelial MODELs, the first EpiOcular ™ (MatTek, MA, USA), HCE (Skin Ethic, france) and CORNEA-modem (labcyto, japen). Wherein, epiOcular of MatTek TM Human epidermal keratinocytes are used as seed cells, and in vitro culture is performed to form a model similar to the corneal epithelial structure, and although the model plays an important role in the evaluation method of ocular-stimulating animal replacement, a non-corneal cell line cannot be completely equivalent to a human cornea. Constructing a CORNEA-MODEL of Labcyte by adopting a normal human CORNEA epithelial cell line, and culturing in vitro to form a CORNEA epithelial structure; corneal epithelial cells are model tools for in vitro studies of cell differentiation, signaling, and cell homeostasis signaling pathways, and can also be used to construct in vitroThree-dimensional corneal epithelial tissue models, however, can be made with limited sources of corneal tissue, combined with a short life cycle of the corneal cells themselves and rapid differentiation, making in vitro culture of the corneal epithelial cells difficult and time consuming. Many attempts to increase the in vitro culture cycle of corneal epithelial cells, such as viral transfection, telomerase reverse transcription transfection, etc., have been made and the HCE model of Skin Ethic is cultured using an immortalized corneal epithelial cell line as seed cells to form a multicellular layer of corneal epithelial tissue analog which is very similar in structure to the corneal mucosa.
The Chinese patent 201410062366 application discloses a simple limbal stem cell separation and in-vitro culture kit and a simple limbal stem cell separation and in-vitro culture method, and the limbal stem cell separation and in-vitro culture method is simple to operate, high in stem cell yield, less in damage to the obtained primary stem cells, strong in proliferation capacity, high in-vitro culture success rate and good in reproducibility.
Chinese patent 03150375.6 discloses a tissue engineering autologous corneal epithelium and a preparation method thereof, wherein the method comprises the steps of amplifying and differentiating corneal epithelial stem cells and corneal epithelial cells derived from a patient in vitro, inoculating the corneal epithelial stem cells and the corneal epithelial cells onto a fibrin biological scaffold for culture, and constructing the tissue engineering autologous corneal epithelium in vitro.
The Chinese patent 200410019341.X application discloses a limbal stem cell tissue engineering complex and a preparation method thereof, wherein the method adopts amniotic membrane with removed epithelium as a carrier, and 3T3 fibroblasts as a trophoblast, so as to construct the tissue engineering cornea complex in vitro.
Chinese patent 200410019342.4 discloses a tissue engineering product of human limbal stem cells nourished by human fibroblasts and a preparation method thereof, wherein the method adopts amniotic membrane with removed epithelium as a carrier, and human fibroblasts are used as a trophoblast to construct a tissue engineering cornea complex in vitro.
The Chinese patent 201019018004.1 application discloses a preparation method of a tissue engineering cornea, which adopts epidermal stem cells and multipotent stromal stem cells as seed cells, and the seed cells are planted on two sides of a decellularized natural cornea stroma after in-vitro amplification culture, and then the tissue engineering cornea is formed through in-vitro induction culture.
The cell sources of the cornea epithelial model include embryonic stem cells, mesenchymal stem cells, skin stem cells, oral mucosa epithelial cells and the like, and animal experiments and clinical applications of the cornea epithelial model as seed cells have been reported and have made breakthrough progress, especially for the research of oral mucosa epithelial cells, which are hot spots at present. In clinical treatment, the method for constructing biological membrane transplantation by using oral mucosa epithelial cells for treating ocular surface diseases is a common method in clinical treatment. Epithelial cells isolated from the oral mucosa are in a lower differentiation stage than epidermal keratinocytes, and because of their relatively short cell cycle, in vitro culture time is also shortened, and prolonged culture time also maintains a non-keratinized state; secondly, residual scars after taking living tissues are not obvious, so that the oral cavity becomes an ideal place for taking the living tissues. Shigeru and Nakamura, etc. indicate that the epithelial tissue of the oral mucosa is composed of 5-6 layers of cells, has basal layers, ratchet layers, granular layers and no horny layer, and has tight connection structures such as desmosomes, semidesmosomes, etc., and the tissue structure is similar to the cornea tissue structure; and the research finds that the oral mucosa epithelial cells express non-stratum corneum specific proteins CK4 and CK13 and cornea specific protein CK3. Studies by Yasutaka et al also indicate that oral mucosal epithelial cells express cornea specific protein CK3, non-stratum corneum specific proteins CK4, CK13, whereas stratum corneum specific proteins CK1, CK10 are not expressed, which is the same as keratin in the cornea.
The successful application of the oral mucosa epithelial cells in clinical treatment shows that the oral mucosa epithelial cells can be used as ideal substitutes of the cornea epithelial cells for ocular surface reconstruction, and the oral mucosa epithelial cells have the potential of replacing cornea cell in-vitro models.
The Chinese patent 200610128698.0 application discloses a preparation method of autologous corneal epithelium, which adopts primary cultured oral mucosa tissue cells of a patient for 3-15 days, carries out passage expansion culture on the obtained cells for 7-30 days, then inoculates the cells on a fibrin biological bracket for culture construction, and finally prepares the autologous corneal epithelium for clinical treatment. However, the cornea model constructed by using the oral mucosa cells at present has long construction time, and a batch of models need to be constructed in vitro for one month, and the uncontrollable factors in the process can be increased to influence the stability of the models due to the overlong construction time; a trophoblast is needed to provide nutrition to the epithelial layer; is applied to clinical treatment.
At present, in vitro construction of tissue engineering cornea is mostly applied to clinical treatment, and bottleneck restricting tissue engineering cornea transplantation is a construction carrier, so research of tissue engineering cornea is mainly focused on the construction carrier, the construction system is more suitable for the carrier, and most of the tissue engineering cornea needs to be nourished for the epithelial layer. In addition, the cornea model constructed by using the cornea cells at present has long construction time, and a batch of models need to be constructed in vitro for one month, and the uncontrollable factors in the process can be increased to influence the stability of the model due to the overlong construction time.
Disclosure of Invention
The embodiment of the invention provides a 3D model constructed in vitro by utilizing a serum-containing culture system and a construction method thereof, and the 3D model has various seed cell selections, good in-vitro construction repeatability and can realize industrialized preparation; meanwhile, a serum-containing culture system is used for providing nutrition required by cells, and meanwhile, the addition of factors is reduced; the construction of the culture system is more suitable for proliferation and differentiation of epithelial cells, so that the cells can be well stratified without a matrix and a bracket, and normal stratification of a model is ensured, thereby forming a connecting structure which is highly similar to human cornea epithelium, and normally expressing cornea epithelium related proteins.
The invention comprises the following steps:
A3D model is a 3D model constructed by utilizing serum-containing culture liquid and a construction method thereof, which applies cell biology and engineering principles, uses primary cornea epithelial cells, limbal stem cells, immortalized cornea epithelial cells, primary oral mucosa epithelial cells, or oral mucosa squamous carcinoma cell line TR146 which are separated by tissues, adopts serum-containing culture liquid and a submerged-gas-liquid level segmented culture method after in-vitro amplification, and forms an in-vitro 3D model.
Further, the cells used are one of primary corneal epithelial cells isolated using tissue, or limbal stem cells, or immortalized corneal epithelial cells, or primary oral mucosal epithelial cells, or oral mucosal squamous carcinoma cell line TR 146.
Further, the culture medium contains 5% -20% of fetal bovine serum FBS.
An in vitro construction method for constructing a 3D model by using serum-containing culture liquid comprises the following method steps:
step one, preparation of cells
Taking primary cornea epithelial cells, or limbal stem cells, or immortalized cornea epithelial cells, or primary oral mucosa epithelial cells, or oral mucosa squamous carcinoma cell line TR146, recovering and amplifying, using P4-P20 generation cells, preparing single cell suspension by pancreatin digestion, and regulating cell density to 5.0X10 4 The volume of the catalyst is between 5.0X10/mL 6 Inoculating to culture flask, adding culture solution I containing 5% -20% foetal calf serum for culturing, gently shaking culture flask to disperse cells uniformly, and placing at 37deg.C and 5% CO 2 Culturing under the condition;
the culture solution I is DMEM, the glucose content of which is 1.0-4.5 g/L, and the glutamine content of which is 0.2-20.0 g/L.
Step two, submerged inoculation culture of 3D model
Taking logarithmic growth phase cells, preparing single cell suspension from culture solution II, and adjusting cell density to 5.0X10 4 The volume of the catalyst is between 5.0X10/mL 6 Inoculating 200 mu L/chamber into a small chamber, placing the small chamber into a model culture dish, adding a proper amount of culture solution II into each dish to ensure that the liquid level heights inside and outside the small chamber are consistent, and performing submerged culture; placing at 37deg.C and 5% CO 2 Culturing for 0.5-2 hours under the condition, transferring into submerged culture for 1-3 days, and changing liquid every day;
the culture solution II is prepared from DMEM: the culture medium with F12 content of 3:1-1:3 is taken as a basic culture medium, fetal bovine serum concentration of 5-20%, retinoic acid concentration of 4-10 mug/mL, human epidermal growth factor concentration of 0.1-10.0 ng/mL, insulin concentration of 5.0-50.0 ng/mL, hydrocortisone concentration of 0.1-5.0 mug/mL, adenine concentration of 5.0-50.0 mug/mL, triiodothyronine concentration of 0.1-10.0 mug/mL, hyaluronic acid concentration of 0.01-0.1%, flavone concentration of 5.0-50.0 mug/mL, glutamine concentration of 0.2 g/L-20.0 g/L, cholera toxin concentration of 1.0-100.0 mug/L, amphotericin B concentration of 1.0-10.0 mg/L, penicillin concentration of 10.0-100.0 IU/mL, streptomycin concentration of 10.0-100.0 mug/mL;
the culture solution II adopted in the steps is added with fetal bovine serum on the basis of basic culture solution, and the fetal bovine serum contains various plasma proteins, polypeptides, fat, carbohydrates, growth factors, hormones, inorganic matters and the like, so that biological factors and physical factors which are lack in artificial synthetic culture solution can be provided, a cell growth place similar to the micro-environment in vivo is formed, and the cell growth or inhibition reaches physiological balance. Moreover, researchers have studied to find that cells cultured in serum-free medium can be well stratified to form a patch only on a matrix or scaffold material, while cells can be well stratified without a matrix or scaffold by adding serum medium. However, some components in serum also have a certain inhibiting effect on epithelial cell differentiation, so various factors are added to the culture solution II to regulate cell proliferation and differentiation. Wherein retinoic acid can promote epithelial cell proliferation, differentiation, and keratolysis. Hydrocortisone may have both cell attachment and proliferation promoting effects and induce differentiation of cells at high cell densities. Hyaluronic acid is a linear polymer polysaccharide composed of glucuronic acid and N-ethyl phthalein glucosamine as disaccharide units, contains a large amount of carboxyl and hydroxyl groups, can form hydrogen bonds with water to combine with a large amount of water, and can regulate the osmotic pressure of a solution; the protective agent has various protective effects on cells, has a blocking effect on the diffusion of charged macromolecules, and has a certain influence on cell behaviors; hyaluronic acid also inhibits the formation and release of free radicals. The flavone contains rich phenolic substances, can be combined with peroxidation free radicals, blocks the progress of peroxidation chain reaction, and improves the antioxidation capability of cells; flavones can also inhibit the occurrence of apoptosis by reducing Caspase-3 protein expression. Aiming at the defect that a single culture medium (such as a DMEM culture medium) used in the traditional culture can only provide amino acid, vitamin, inorganic salt, organic compound and trace element, the addition of the factors ensures the nutrition requirement of cells and promotes the growth of the cells; on the other hand, the activity of the cells is ensured in terms of osmotic pressure, antioxidant capacity and the like of the culture medium.
Step three, gas-liquid surface proliferation and differentiation culture of 3D model
Discarding the culture solution in the small chamber, replacing the culture solution in the model culture dish with culture solution III to ensure that the liquid level and the upper surface of the cells in the small chamber are positioned on the same horizontal line, performing gas-liquid level culture for 1-7 days, changing the liquid every day, finishing the culture, and finishing the in vitro construction of the model;
the culture solution III is prepared by adding hydrocortisone with a concentration of 5.0-10.0 mug/mL, hyaluronic acid with a concentration of 0.1-1%, flavone with a concentration of 50.0-150.0 mug/mL and vitamin E with a concentration of 20.0-80.0 mug/mL into the culture solution II.
In the steps, a gas-liquid level sectional culture method is adopted to make the gas-liquid level sectional culture be stratified to form an in-vitro model. The culture solution III used in the gas-liquid surface culture stage is different from the culture solution II in that the concentration of hydrocortisone, hyaluronic acid and flavone in the culture solution II is increased, and vitamin E is added. Vitamin E is one of the most important antioxidants, protects cells from poisoning by free radicals, and inhibits apoptosis; the oxygen demand of cells can be reduced, and the activity of the cells can be maintained; and are involved in the synthesis of cellular DNA. The proper increase of the hydrocortisone concentration can play a role in inducing cell differentiation under the condition of increasing the cell density, and ensure complete stratification of the model. Hyaluronic acid inhibits the formation and release of free radicals on the one hand and maintains osmotic pressure required by normal metabolism of cells on the other hand, thereby ensuring proliferation and differentiation of cells. The increase in flavone concentration further inhibits apoptosis of cells during rapid growth. The combined action of the conditions ensures the rapid and healthy proliferation and differentiation of cells, finally forms a stratified cornea epithelial structure, simplifies the construction method and shortens the construction time.
Further, the construction time of the model is 3-10 days, and the construction time is short.
Further, the construction system can provide nutrition and microenvironment required by proliferation and differentiation of epithelial cells, and a trophoblast is not required.
Further, the model structure is similar to the normal human cornea epithelium structure, and is a multi-layered structure without horny layer.
Further, the model cornea specific protein expressed similar to normal human cornea epithelium, expressing cornea specific protein CK3.
Further, the barrier function was examined by the ET50 method (time required for half of the cells in the model to die by 0.3% Triton X-100), and the ET50 was 5 to 15min after completion of submerged culture.
Further, the barrier function is detected by using an ET50 method, and after the gas-liquid surface culture is finished, the ET50 is 30-70 min.
Further, the model is applied to in vitro detection of eye irritation of products such as small molecular compounds, active proteins, medical instruments, chemicals, cosmetics and the like.
The beneficial effects of the invention are as follows:
the 3D model constructed by the method provided by the invention has the following advantages: the seed cells are selected variously, the in vitro construction repeatability is good, and the industrialized preparation can be realized; 2. the serum-containing culture system is used for providing nutrition required by cells and reducing the addition of factors; thirdly, the addition of hyaluronic acid can inhibit the formation and release of free radicals, can regulate the osmotic pressure of the solution on the one hand and can maintain the osmotic pressure required by normal metabolism of cells, thereby ensuring proliferation and differentiation of the cells; 4. the addition of flavone reduces the expression of Caspase-3 protein, thereby inhibiting the occurrence of apoptosis; 5. different hydrocortisone concentrations in the submerged and gas-liquid surface stages ensure the requirements of cells in different periods and are beneficial to the formation of a stratified cornea epithelial structure; 6. the gas-liquid surface culture stage is simplified, so that the construction time is shortened and the production cost is reduced while the rapid proliferation and differentiation of cells are ensured; seventhly, the constructed culture system is more suitable for proliferation and differentiation of epithelial cells, so that the cells can be well stratified without a matrix and a bracket, normal stratification of a model is ensured, a connecting structure which is highly similar to human cornea epithelium is formed, cornea epithelium related proteins are normally expressed, the barrier function of the model can be further improved under the influence of the self structure and extracellular microenvironment, and finally, the constructed in vitro 3D model has highly similar structure and function with human cornea epithelium tissue; eighth, use 3D model that is set up to detect some chemicals in the unified classification and label system of chemical all over the world, the result shows, 3D model that the method that the invention provides prepared can judge accurately whether this chemical has eye irritation, its result is identical with judgement result of rabbit eye experiment, therefore, 3D model can reflect the detection result objectively and truly, can very good substitute animal model, become the core tool of the in vitro test, apply to eye irritation in vitro detection of products such as small molecule compound, active protein, medical instrument, chemicals, cosmetics, etc..
Drawings
FIG. 1 is a photograph of a histological section HE stained with a 3D model constructed outside of serum-containing culture fluid;
FIG. 2 is a photograph of immunohistochemical staining of keratin CK3 using a 3D model constructed outside serum-containing culture fluid;
FIG. 3 is a graph of tissue activity versus 0.3% Triton X-100 action time after completion of submerged culture using a 3D model constructed outside of serum-containing culture fluid, and calculated ET50 values;
FIG. 4 is a graph of tissue activity versus 0.3% Triton X-100 action time after completion of gas-liquid surface culture using a 3D model constructed outside of serum-containing culture fluid, and calculated ET50 values;
FIG. 5 is a graph showing the results of compound eye irritation detection using a 3D model constructed outside serum-containing culture fluid.
Detailed Description
The invention is further illustrated by the following examples of embodiments, which are not intended to limit the claims.
Example 1:
this example focuses on the in vitro construction method steps of a 3D model constructed in vitro using primary corneal epithelial cells:
step one, preparation of cells
Taking primary cornea epithelial cells, resuscitating and amplifying, preparing single cell suspension by using P7 generation cells through pancreatin digestion, and regulating the cell density to 5.0x10 4 Inoculating to culture flask at a volume of one mL, adding 10% fetal bovine serum culture solution I, culturing, slightly shaking culture flask to disperse cells uniformly, and standing at 37deg.C in 5% CO 2 Culturing under the condition;
the culture solution I is DMEM, the glucose content of the culture solution I is 1.0g/L, and the glutamine content of the culture solution I is 0.2g/L.
Step two, submerged inoculation culture of 3D model
Taking logarithmic growth phase cells, preparing single cell suspension from culture solution II, and adjusting cell density to 5.0X10 4 Inoculating 200 mu L/chamber into a small chamber, placing the small chamber into a model culture dish, adding a proper amount of culture solution II into each dish to ensure that the liquid level heights inside and outside the small chamber are consistent, and performing submerged culture; placing at 37deg.C and 5% CO 2 Culturing for 0.5 hr, transferring into submerged culture for 3 days, and changing liquid every day;
the culture solution II is prepared from DMEM: the culture medium with F12 content of 3:1 is taken as a basic culture medium, and added with fetal bovine serum with concentration of 20%, retinoic acid with concentration of 8 mug/mL, human epidermal growth factor with concentration of 0.8ng/mL, insulin with concentration of 20.0 ng/mL, hydrocortisone with concentration of 0.5 mug/mL, adenine with concentration of 25.0 mug/mL, triiodothyronine with concentration of 0.1 mug/mL, hyaluronic acid with concentration of 0.1%, flavone with concentration of 10.0 mug/mL, glutamine with concentration of 0.2g/L, cholera toxin with concentration of 1.0 mug/L, amphotericin B with concentration of 1.0mg/L, penicillin with concentration of 100.0IU/mL, streptomycin with concentration of 100.0 mug/mL;
step three, gas-liquid surface proliferation and differentiation culture of 3D model
Discarding the culture solution in the small chamber, replacing the culture solution in the model culture dish with culture solution III to ensure that the liquid level and the upper surface of the cells in the small chamber are positioned on the same horizontal line, performing gas-liquid level culture for 7 days, changing the culture solution every day, and finishing the in-vitro construction of the 3D model;
in the culture solution III, the concentration of hydrocortisone is increased to 10.0 mug/mL, the concentration of hyaluronic acid is 1%, the concentration of flavone is 100.0 mug/mL, and the concentration of vitamin E is added to 50.0 mug/mL.
The results of histological section HE staining of the 3D model constructed in vitro using primary corneal epithelial cells are shown in fig. 1, illustrating the integrity of the 3D model stratified structure constructed in vitro using primary corneal epithelial cells.
Example 2:
the present example focuses on the in vitro construction method steps of a 3D model constructed in vitro using immortalized corneal epithelial cells:
step one, preparation of cells
Collecting immortalized corneal epithelial cells, resuscitating and amplifying, preparing single cell suspension by using P20 generation cells, and regulating cell density to 5.0X10 5 Inoculating to culture flask at a volume of one mL, adding 10% fetal bovine serum culture solution I, culturing, slightly shaking culture flask to disperse cells uniformly, and standing at 37deg.C in 5% CO 2 Culturing under the condition;
the culture solution I is DMEM, the glucose content of the culture solution I is 4.5g/L, and the glutamine content of the culture solution I is 0.2g/L.
Step two, submerged inoculation culture of 3D model
Taking logarithmic growth phase cells, preparing single cell suspension from culture solution II, and adjusting cell density to 5.0X10 5 Inoculating 200 mu L/chamber into a small chamber, placing the small chamber into a model culture dish, adding a proper amount of culture solution II into each dish to ensure that the liquid level heights inside and outside the small chamber are consistent, and performing submerged culture; placing at 37deg.C and 5% CO 2 Culturing for 2 hours under the condition, transferring into submerged culture for 3 days, and changing liquid every day;
the culture solution II is prepared from DMEM: the culture medium with F12 content of 3:1 is taken as a basic culture medium, and added with fetal bovine serum with concentration of 20%, retinoic acid with concentration of 8 mug/mL, human epidermal growth factor with concentration of 0.5ng/mL, insulin with concentration of 20.0 ng/mL, hydrocortisone with concentration of 0.5 mug/mL, adenine with concentration of 25.0 mug/mL, triiodothyronine with concentration of 0.1 mug/mL, hyaluronic acid with concentration of 0.1%, flavone with concentration of 8.0 mug/mL, glutamine with concentration of 0.2g/L, cholera toxin with concentration of 1.0 mug/L, amphotericin with concentration of 1.0mg/L, penicillin with concentration of 100.0IU/mL, streptomycin with concentration of 100.0 mug/mL;
step three, gas-liquid surface proliferation and differentiation culture of 3D model
Discarding the culture solution in the small chamber, replacing the culture solution in the model culture dish with culture solution III to ensure that the liquid level and the upper surface of the cells in the small chamber are positioned on the same horizontal line, performing gas-liquid level culture for 7 days, changing the culture solution every day, and finishing the in-vitro construction of the 3D model;
in the culture solution III, hydrocortisone with the concentration of 8.0 mug/mL, hyaluronic acid with the concentration of 0.5% and flavone with the concentration of 80.0 mug/mL and vitamin E with the concentration of 40.0 mug/mL are added.
The results of immunohistochemical staining of keratin CK3 using the 3D model constructed in vitro with immortalized corneal epithelial cells are shown in fig. 2, illustrating that the protein expression of the 3D model constructed in vitro with immortalized corneal epithelial cells is similar to human cornea.
Example 3:
the present example focuses on the in vitro construction method steps of a 3D model constructed in vitro using oral mucosa squamous cell carcinoma cell line TR 146:
step one, preparation of cells
Taking oral mucosa squamous cancer cell strain TR146, resuscitating and amplifying, using P14 generation cells, preparing single cell suspension by pancreatin digestion, and regulating cell density to 5.0X10 6 Inoculating to culture flask at a volume of one mL, adding 10% fetal bovine serum culture solution I, culturing, slightly shaking culture flask to disperse cells uniformly, and standing at 37deg.C in 5% CO 2 Culturing under the condition;
the culture solution I is DMEM, the glucose content of the culture solution I is 1.0g/L, and the glutamine content of the culture solution I is 0.2g/L.
Step two, submerged inoculation culture of 3D model
Taking logarithmic growth phase cells, preparing single cell suspension from culture solution II, and adjusting cell density to 5.0X10 6 Inoculating 200 mu L/chamber into a small chamber, placing the small chamber into a model culture dish, adding a proper amount of culture solution II into each dish to ensure that the liquid level heights inside and outside the small chamber are consistent, and performing submerged culture; placing at 37deg.C and 5% CO 2 Culturing for 2 hours under the condition, transferring into submerged culture for 2 days, and changing liquid every day;
the culture solution II is prepared from DMEM: the culture medium with F12 content of 3:1 is taken as a basic culture medium, the concentration of fetal bovine serum is 10%, the concentration of retinoic acid is 8 mug/mL, the concentration of human epidermal growth factor is 0.5ng/mL, the concentration of insulin is 30.0 ng/mL, the concentration of hydrocortisone is 1.0 mug/mL, the concentration of adenine is 50.0 mug/mL, the concentration of triiodothyronine is 0.1 mug/mL, the concentration of hyaluronic acid is 0.3%, the concentration of flavone is 8.0 mug/mL, the concentration of glutamine is 0.2g/L, the concentration of cholera toxin is 1.0 mug/L, the concentration of amphotericin B is 1.0mg/L, the concentration of penicillin is 100.0IU/mL, and the concentration of streptomycin is 100.0 mug/mL;
step three, gas-liquid surface proliferation and differentiation culture of 3D model
Discarding the culture solution in the small chamber, replacing the culture solution in the model culture dish with culture solution III to ensure that the liquid level and the upper surface of the cells in the small chamber are positioned on the same horizontal line, performing gas-liquid level culture for 6 days, changing the culture solution every day, and finishing the in-vitro construction of the 3D model;
in the culture solution III, hydrocortisone with the concentration of 8.0 mug/mL, hyaluronic acid with the concentration of 0.6%, flavone with the concentration of 100.0 mug/mL and vitamin E with the concentration of 50.0 mug/mL are added.
Example 4:
this example focuses on the procedure for ET50 detection using a 3D model constructed in vitro of primary corneal epithelial cells:
1) The model was placed in a 6-well plate, and 0.9. 0.9mL of culture medium was added to the plate.
2) mu.L of 0.3% Triton X-100 was pipetted and slowly dropped onto the tissue surface to ensure that the reagent was as covering the tissue surface as possible.
3) The administration time is 0, 30 and 60min respectively.
4) After the last tissue dose, all 6-well plates were transferred to a constant temperature incubator (37.+ -. 1 ℃ C., 5.+ -. 1% CO) 2 95% relative humidity).
5) 30min before tissue incubation was completed, 1mg/mL MTT solution was prepared and 300. Mu.L of MTT solution was added to each well of the 24-well plate.
6) After the end of the administration, the washing procedure was started, and the tissue was washed with a wash bottle containing sterile DPBS, and after 10 times, the tissue culture chambers were immersed in DPBS, once each.
7) The washed and dried tissue was transferred to a 24-well plate with MTT test solution added thereto, and the tissue was incubated in a constant temperature incubator (37.+ -. 1 ℃ C., 5.+ -. 1% CO 2 95% relative humidity), incubate 3 hmin.
8) After the MTT incubation was completed, the MTT solution was gently aspirated from the well plate, and DPBS solution was added to the well plate for the washing process.
9) After washing, the tissue bottom surface was wiped dry with absorbent paper, transferred to a new 24-well plate, and 2mL isopropanol was added to the culture chamber, which was able to dissolve the crystals produced by MTT. And a sealing film is adopted to seal a gap of the 24 pore plates, so that the final volume is prevented from being influenced by volatilization of isopropanol. Standing at 4 ℃ overnight for dissolution.
10 After completion of the lysis, 2 parts of 200 μl of purple formazan solution were pipetted into the same 96-well plate for each tissue. Parallel tissues were treated repeatedly and fluid was transferred according to the design of the well plate, using isopropanol as a blank. The spectrophotometer 570nm wavelength reads absorbance without the use of a filter.
11 FIG. 3 shows a graph of tissue activity versus 0.3% Triton X-100 action time after completion of submerged culture in 3D model, and the ET50 value was calculated to be 11.7min. The graph of tissue activity after the end of the gas-liquid surface culture in the 3D model and the action time of 0.3% Triton X-100 is shown in FIG. 4, and the ET50 value is calculated to be 59.6min.
Example 5:
this example focuses on the procedure for compound eye irritation detection using 3D models constructed in vitro of immortalized corneal epithelial cells:
1) Preparing a sample
The experimental group is carried out, wherein the sample group is 2 liquid chemical standard substances, namely dipropyldisulfide and N, N-diethyl m-toluidine, the dipropyldisulfide belongs to non-ocular surface stimulating chemical substances in human body experiments, the human body experimental result of the N, N-diethyl m-toluidine is ocular surface stimulating chemical substances, the negative control group is ultrapure water, and the positive control group is methyl acetate.
2) Testing security
(1) 1 six well plates were prepared for each test article, and a total of 46 well plates were prepared. 0.9ml of DMEM cell culture medium was added to each well of the first row, and each well was placed with a surface area of 0.5cm 2 In vitro recombinant corneal epithelial model(s).
(2) Treating the surface of the tissue in step (1) with 20. Mu.L of Du's phosphate buffer before the test and control substances are treated, and treating the surface with 5% CO at 36-37 ℃ in a dark environment 2 The cells were incubated in an incubator with 95% relative humidity (standard incubation conditions) for 15min to simulate the wet state of the human eye.
(3) The test substance is administered. All dosing operations were performed in an ultra clean bench: every 30s a model is administered (the time may be variable, controlled by the self administration time, but it is ensured that the administration time of each model is constant), ensuring that a sufficient soak time interval remains after administration. At 0.5cm 2 The administration was performed on the tissue surfaces, respectively, and the administration amount was 83.3. Mu.L/cm 2 (liquid chemical) or 83.3mg/cm 2 After administration (solid chemicals), the cell chamber was gently swirled to promote spreading of the test substance on the tissue surface and incubated for 30min at room temperature. Note that: the tissue surface is not compressible.
(4) At the end of the time of administration to the first administered tissue, the in vitro recombinant corneal epithelial model was removed and three sterile 24-well plates were prepared for cell viability assays.
(5) The washing procedure is started. Each culture chamber was controlled to wash for 30s, the in vitro recombinant corneal epithelium model was washed 15 times with Dunaliella salt buffer, and rubbed with sterile gauze or sterile cotton stick.
(6) After the cleaning is finished, the mixture is washed at 36 to 37 ℃ and 5 percent CO 2 The tissue was cultured using fresh DMEM medium under 95% relative humidity culture conditions for 120min.
(7) MTT test surface-dried in vitro recombinant corneal epithelium model was transferred to 24 well plates containing 1mg/mL thiazole blue solution, 0.3mL per well, at 36-37℃with 5% CO 2 Incubate in a 95% humidity cell incubator for 3 hours in the dark.
(8) The thiazole blue solution is sucked by a pipette, the in-vitro recombined cornea epithelial model is completely immersed into a 24-orifice plate containing 2mL of isopropanol in each orifice, the 24-orifice plate is sealed by a sealing film, and the mixture is stood and extracted at the temperature of 4 ℃ for 12 to 16 hours, so that the extract of the isopropanol is obtained.
(9) The bottom of the culture chamber was pierced with a 200. Mu.l pipette tip and the isopropanol extract of the in vitro reconstituted corneal epithelial model was allowed to flow into the culture well. 200 μl of the isopropanol extract was pipetted into a 96-well plate.
(10) Measuring absorbance: the absorbance OD at a wavelength of 550-570 nm was measured with a 96-well plate spectrophotometer.
(11) Determining the irritation: the absorbance value of negative control deionized water is taken as denominator, the absorbance values of test sample dipropyldisulfide and N, N-diethyl m-toluidine are taken as molecules respectively, and the percentage of the ratio is taken as the respective relative cell viability. The results are shown in FIG. 5, where dipropyldisulfide is present at a relative 69.1% and relative cell viability is higher than 60% and therefore is a non-ocular surface stimulating chemical. The relative cell activity of N, N-diethyl m-toluidine is 20.7%, and is lower than 60%, which indicates that N, N-diethyl m-toluidine has irritation to ocular surface. (relative cell viability above 60%, the chemical tested was non-ocular surface irritating, relative cell viability below 60%, the chemical tested was irritating.)
While the basic principles and main features of the present invention and advantages of the present invention have been shown and described, it will be understood by those skilled in the art that the present invention is not limited by the foregoing embodiments, which are described in the foregoing specification merely illustrate the principles of the present invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention, which is defined in the appended claims and their equivalents.

Claims (6)

1. An in vitro construction method for constructing a 3D model by using a serum-containing culture liquid is characterized by comprising the following method steps of:
step one, preparation of cells
Taking primary corneal epithelial cells, or limbal stem cells, or immortalized corneal epithelial cells, or primary oral mucosa epithelial cells, or oral mucosa squamous carcinoma cell line TR146, recovering and amplifying, using P4-P20 generation cells, preparing single cell suspension by pancreatin digestion, and regulating cell density to 5.0X10 4 The volume of the catalyst is between 5.0X10/mL 6 Inoculating to culture flask, culturing in culture solution I containing 5-20% fetal bovine serum, slightly shaking culture flask to disperse cells uniformly, and standing at 37deg.C and 5% CO 2 Culturing under the condition;
the culture solution I is DMEM, the glucose content of which is 1.0-4.5 g/L, and the glutamine content of which is 0.2-20.0 g/L;
step two, submerged inoculation culture of 3D model
Taking logarithmic growth phase cells, preparing single cell suspension from culture solution II, and adjusting cell density to 5.0X10 4 The volume of the catalyst is between 5.0X10/mL 6 Inoculating 200 mu L/chamber into a small chamber, placing the small chamber into a model culture dish, adding a proper amount of culture solution II into each dish to ensure that the liquid level heights inside and outside the small chamber are consistent, and performing submerged culture; placing at 37deg.C and 5% CO 2 Culturing for 0.5-2 hours under the condition, transferring into submerged culture for 1-3 days, and changing liquid every day;
the culture solution II is prepared from DMEM: the culture medium with F12 content of 3:1-1:3 is taken as a basic culture medium, fetal bovine serum concentration of 5% -20%, retinoic acid concentration of 4-10 mug/mL, human epidermal growth factor concentration of 0.1-10.0 ng/mL, insulin concentration of 5.0-50.0 ng/mL, hydrocortisone concentration of 0.1-5.0 mug/mL, adenine concentration of 5.0-50.0 mug/mL, triiodothyronine concentration of 0.1-10.0 mug/mL, hyaluronic acid concentration of 0.01% -0.1%, flavone concentration of 5.0-50.0 mug/mL, glutamine concentration of 0.2 g/L-20.0 g/L, cholera toxin concentration of 1.0-100.0 mug/L, amphotericin B concentration of 1.0-10.0 mg/L, penicillin concentration of 10.0-100.0 IU/mL, streptomycin concentration of 10.0-100.0 mug/mL;
step three, gas-liquid surface proliferation and differentiation culture of 3D model
Discarding the culture solution in the small chamber, replacing the culture solution in the model culture dish with culture solution III to ensure that the liquid level and the upper surface of the cells in the small chamber are positioned on the same horizontal line, performing gas-liquid level culture for 1-7 days, changing the liquid every day, finishing the culture, and finishing the in vitro construction of the model;
the culture solution III is prepared by adding hydrocortisone with a concentration of 5.0-10.0 mug/mL, hyaluronic acid with a concentration of 0.1-1%, flavone with a concentration of 50.0-150.0 mug/mL and vitamin E with a concentration of 20.0-80.0 mug/mL into the culture solution II.
2. The method for constructing a 3D model in vitro using serum-containing culture fluid according to claim 1, wherein the model construction time is 3 to 10 days.
3. The method according to claim 1 or 2, wherein the barrier function of the 3D model constructed outside the serum-containing culture liquid is measured by the ET50 method, and the time required for half of the cells in the model to die after completion of submerged culture is 5 to 15 minutes with ET50 of 0.3% triton x-100.
4. The method for constructing a 3D model in vitro using a serum-containing culture liquid according to claim 1 or 2, wherein the barrier function of the 3D model constructed in vitro using a serum-containing culture liquid is detected by an ET50 method, and the ET50 is 30 to 70min after the completion of the air-liquid surface culture.
5. The in vitro construction method for constructing a 3D model using serum-containing culture liquid according to claim 1 or 2, wherein the model is applied to in vitro detection of eye irritation of small molecule compounds, active proteins, medical devices, chemicals, cosmetics and the like.
6. A 3D model constructed according to the method of claims 1-5.
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