CN114540275A - Skin biological printing ink and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a skin biological printing ink, relating to the technical field of skin tissue engineering, comprising a 50-200 mug/mL laminin solution, wherein the laminin solution contains gelatin with the mass concentration of 3-10% and sodium alginate with the mass concentration of 0.5-2%. The invention also provides a preparation method and application of the skin biological printing ink. The invention has the beneficial effects that: the laminin solution contains 3-10% of gelatin and 0.5-2% of sodium alginate by mass concentration to form skin biological printing ink, which is beneficial to proliferation of cells in a dermis layer, the survival rate of the cells in the dermis layer is high, and growth and differentiation of cells in an epidermis layer are facilitated when a full-layer skin is further formed in the later stage, so that a compact and thicker epidermis layer is formed.
Description
Technical Field
The invention relates to the technical field of skin tissue engineering, in particular to skin biological printing ink and a preparation method and application thereof.
Background
The skin is the largest organ of the human body and plays an important role in protecting the human body from the external environment. Since the skin is the first line of defense in the human body, it is susceptible to various types of injury (e.g., traumatic wounds, burns, diabetic feet, etc.). When the skin is seriously damaged and can not repair itself, the current main treatment method is autografting or tissue engineering skin auxiliary treatment. Due to the limited transplantable autologous skin, the development of tissue engineering skin substitutes for the treatment of skin lesions is of great importance. Furthermore, european legislation prohibits the use of animals for testing cosmetic ingredients and increases the number of industrial chemicals that must be risk assessed. Therefore, there is also a strong need in the cosmetic and pharmacological fields for reliable, high quality, reproducible skin substitutes to replace animals for testing.
Bioprinting techniques have been developed vigorously in the field of bio-fabrication, which can manipulate the spatial distribution of cells and extracellular matrix, mimicking the biological structure of a tissue or organ. The rapid development of bioprinting technology provides a new idea for tissue engineered skin construction, and several researchers have now demonstrated the potential of bioprinting skin tissues, which have evolved from simple to complex, low to high biomimetic. Lee et al led to the printing of skin tissue based on a Multi-layer build-up method (collagen-fibroblast-collagen-keratinocyte-collagen) using bioprinting techniques (Lee, W., et al, Multi-layered culture of human skin fibers and tissue microorganisms, biomaterials,2009.30(8): p.1587-95.); binder et al used a swine wound model with a defective back skin for the first time to perform in vivo bioprinting, and directly printed the bio-ink containing cells onto the damaged skin surface based on inkjet bioprinting, and the results showed that the epithelization rate in the printed group was significantly higher than that in the control group (Binder KW, ZHao W, Aboushwareb T, et al. in situ bioprinting of the skin for burns. J Am Coll Surg 2011; 211: S76.). Although there have been some advances in biological printing of skin, currently printed skin is not widely used, mainly because the current skin printing technology is complicated to operate and is not suitable for preparing skin samples in batches, and in addition, the current printed skin samples have low imitativeness and reduced applicability.
A key factor in 3D printing skin is the raw material used for 3D bioprinting, i.e. bio-ink. Constructing an in vitro skin model or skin disease model can be used for studying the mechanisms of skin diseases, skin irritation tests, anti-aging compound tests, and the like. However, at present, building a personalized and customized full-thickness skin (including epidermis layer and dermis layer) model based on a bioprinting technology is still in a primary stage, and the current preparation method is relatively complex in operation, relatively high in cost, difficult to prepare in batches, difficult to form a compact epidermis layer, and not well applicable to subsequent research. Patent application publication No. CN109054503A discloses a method for preparing a bioprinting ink, a printing method and application thereof, but the bioprinting ink is not suitable for skin bioprinting.
Disclosure of Invention
One of the technical problems to be solved by the present invention is to provide a bioprinting ink suitable for skin.
The invention solves the technical problems through the following technical means:
a skin biological printing ink comprises 50-200 mug/mL of laminin solution, wherein the laminin solution contains 3-10% of gelatin and 0.5-2% of sodium alginate by mass concentration.
Has the advantages that: the laminin solution contains 3-10% of gelatin and 0.5-2% of sodium alginate by mass concentration to form the skin biological printing ink, which is beneficial to the proliferation of cells in a dermis layer, has high survival rate of the cells in the dermis layer, and is beneficial to the growth and differentiation of cells in an epidermis layer when a full-layer skin is further formed in the later stage, so that a compact and thick epidermis layer is formed.
The preparation method of the skin biological printing ink comprises the following steps:
(1) placing sodium alginate powder and gelatin powder under ultraviolet lamp for irradiation, adding DMEM complete culture medium, dissolving, and sterilizing to obtain mixture of sodium alginate and gelatin;
(2) and (3) adding the mixture of sodium alginate and gelatin in the step (1) into the laminin solution to form the skin biological printing ink.
Has the advantages that: according to the invention, gelatin with the mass concentration of 3-10% and sodium alginate with the mass concentration of 0.5-2% are added into the fibronectin solution to form the skin biological printing ink, which is beneficial to the proliferation of cells in a dermis layer, the cell survival rate is very high, and the growth and differentiation of cells in an epidermis layer are facilitated when a full-layer skin is further formed in the later stage, so that a compact and thick epidermis layer is formed.
Preferably, the cultured dermal cells are trypsinized, the cells are suspended in a laminin solution, and then a mixture of sodium alginate and gelatin is added.
Preferably, the final concentration of the dermal layer cells is 5 × 106/ml。
The second technical problem to be solved by the invention is that the existing skin printing biological ink is not beneficial to the attachment of the epidermal layer, so that a complete and compact epidermal layer cannot be formed, and the invention provides a method for preparing a full-layer skin model by adopting the skin biological printing ink.
A method for preparing a full-thickness skin model by using skin biological printing ink comprises the following steps:
(1) placing sodium alginate powder and gelatin powder under ultraviolet lamp for irradiation, adding DMEM complete culture medium, dissolving, and sterilizing to obtain mixture of sodium alginate and gelatin;
(2) digesting the cultured dermal layer cells by trypsin, suspending the cells by a laminin solution, and then adding a mixture of sodium alginate and gelatin to form biological printing ink containing the dermal layer cells;
(3) after the dermal layer cell biological printing ink is cooled to gel, placing the gel in a charging barrel of a 3D printer, setting a dermal layer printing model, and extruding the ink for printing to form a dermal layer model;
the dermis layer model comprises a first ink layer, a second ink layer, a third ink layer and a fourth ink layer, wherein the first ink layer, the second ink layer and the third ink layer are sequentially laminated, the fourth ink layer encloses the first ink layer, the second ink layer and the third ink layer, and a funnel-shaped space with a plurality of gradually-increased openings is formed between the second ink layer and the third ink layer;
(4) and (2) crosslinking the printed dermis model into gel, cleaning, placing on a porous support, placing the support in a culture dish, paving a laminin solution on the upper surface of the third ink layer, placing in an incubator for incubation, paving a epithelial cell suspension, performing static culture in the incubator, adding an epidermal differentiation culture medium into the culture dish until the part below the epidermal cell suspension, and performing gas-liquid phase culture to form the full-layer skin model.
Has the advantages that: according to the invention, gelatin with the mass concentration of 3-10% and sodium alginate with the mass concentration of 0.5-2% are added into the fibronectin solution to form the skin biological printing ink, so that the proliferation of cells in a dermis layer is facilitated, the cell survival rate is high, and the attachment of the epidermis layer is facilitated, so that a complete and compact epidermis layer is formed.
The dermal layer cell biological printing ink can be extruded for printing after being cooled to be colloid, and can not be printed if being cooled to be liquid, and the printing ink can be extruded in a dripping shape.
The traditional gas-liquid phase culture uses a commercial Trans well, but the vessel has very high cost and very limited capacity of a storage culture medium, so that the culture medium needs to be frequently replaced in the culture process, and the invention occupies the precious energy of scientific research personnel.
Preferably, in the step (4), the dermis model is placed into a calcium chloride aqueous solution with the mass concentration of 2% to be crosslinked into gel.
Preferably, the first ink layer comprises two transversely and longitudinally oriented layers, the first ink layer having a line spacing of 0 mm.
Preferably, the second ink layer comprises two layers running transversely and longitudinally, the line spacing between each of the second ink layers being 0.8 mm.
Preferably, the third ink layer comprises two transversely and longitudinally running layers, the line spacing between each of the second ink layers being 1.6 mm.
Preferably, the porous scaffold is a stainless steel mesh or a plastic scaffold.
Has the advantages that: the stainless steel net or the plastic bracket has very low cost and can be repeatedly used after being sterilized.
Preferably, the laminin solution in the step (3) has a concentration of 100 μ g/mL.
Preferably, the concentration of the epidermal cell suspension in the step (3) is 2 × 107/ml。
The invention has the advantages that: the laminin solution contains 3-10% of gelatin and 0.5-2% of sodium alginate by mass concentration to form the skin biological printing ink, which is beneficial to the proliferation of cells in a dermis layer, has high survival rate of the cells in the dermis layer, and is beneficial to the growth and differentiation of cells in an epidermis layer when a full-layer skin is further formed in the later stage, so that a compact and thick epidermis layer is formed.
According to the invention, gelatin with the mass concentration of 3-10% and sodium alginate with the mass concentration of 0.5-2% are added into the fibronectin solution to form the skin biological printing ink, so that the proliferation of cells in a dermis layer is facilitated, the cell survival rate is high, and the attachment of the epidermis layer is facilitated, so that a complete and compact epidermis layer is formed.
The traditional gas-liquid phase culture uses a commercial Trans well, but the vessel has very high cost and very limited capacity of a storage culture medium, so that the culture medium needs to be frequently replaced in the culture process, and the invention occupies the precious energy of scientific research personnel.
The stainless steel net bracket has very low cost and can be used repeatedly after being sterilized.
Drawings
FIG. 1 is a morphological diagram of cells in ink according to example 1, example 2 and comparative example 1 of the present invention; in the figure, A is epidermal cells obtained by digestion in example 2, B is epidermal cells obtained by digestion in example 2 and grown in ink, C is epidermal cells obtained by digestion in example 2 and grown in ink without laminin, D is dermal cells obtained by digestion in example 1, E is dermal cells obtained by digestion in example 1 and F is dermal cells obtained by digestion in ink without laminin in example 1;
FIG. 2 is a graph of a cell survival assay of the present invention; in the figures, A-C show the patterns (0.33mm,0.41mm,0.51mm) printed with nozzles of different sizes; D-F represents the corresponding cell survival assay after 7 days of culture;
FIG. 3 is a schematic diagram of a dermal layer model according to embodiment 5 of the present invention; in the figure, A is the first ink layer, B is the first ink layer, the second ink layer and part of the fourth ink layer, and C is the integral model of the dermis layer;
FIG. 4 is a diagram illustrating a real object of a printing process of a dermis layer model in embodiment 5 of the present invention;
FIG. 5 is a diagram of the dermal layer model printing process and the gas-liquid culture in example 5 of the present invention;
FIG. 6 is a graph of full-thickness skin HE staining results obtained in example 5 of the present invention; in the figure, A is the HE staining result of human skin tissue, and B-D are the HE staining results of different days of printed skin tissue culture.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The specific techniques or conditions not specified in the examples can be performed according to the techniques or conditions described in the literature in the field or according to the product specification.
Example 1
The preparation of the biological printing ink containing the dermal layer cells comprises the following steps:
(1) placing sodium alginate powder and gelatin powder under an ultraviolet lamp for irradiation for 30min, adding a DMEM complete culture medium, wherein the final concentration of sodium alginate is 2% (wt%), and the final concentration of gelatin is 10% (wt%), then placing the suspension in a 60 ℃ oven for 1h to fully dissolve the sodium alginate and the gelatin, after the mixture of the sodium alginate and the gelatin is cooled to room temperature, placing the mixture in the 60 ℃ oven for pasteurization for 1h to obtain a mixture of the sodium alginate and the gelatin, and placing the mixture in a cell culture box for later use;
(2) cell culture: firstly, sucking out original culture medium in a culture dish by using a pipette; then, washing with PBS for 3 times, removing residual culture medium, adding 2mL of pancreatin (0.25% Trypsin-EDTA), placing the mixture back to the incubator for digestion for about 3min, and adding 2mL of DMEM complete culture medium to stop digestion when the cells become round and part of the cells float; finally, the cells were gently pipetted off the bottom of the dish, the liquid was then transferred to a centrifuge tube and centrifuged in a centrifuge (120g,5min), the supernatant was discarded and fresh medium was added for resuspension.
(2) Cell resuspension was performed with 100 μ g/ml laminin solution (solvent was normal saline), and then the cell suspension containing laminin was mixed with sodium alginate-gelatin mixture 1: 1 to form the final bioprinting ink containing dermal cells, the final concentrations of the ink sodium alginate, gelatin, laminin and its cells being: 1% (w/v), 5% (w/v), 50. mu.g/ml and 5X 106/ml。
Example 2
This embodiment is different from embodiment 1 in that: the cells of the dermis layer are replaced by the cells of the epidermis.
Comparative example 1
This embodiment is different from embodiment 1 in that: the laminin solution was replaced with an equal amount of physiological saline.
Comparative example 2
This embodiment is different from embodiment 1 in that: the cells of the dermis layer were replaced with the cells of the epidermis, while the laminin solution was replaced with an equal amount of physiological saline.
Fig. 1 shows the morphology of cells in the bio-printing ink, and it can be seen that when laminin is contained in the bio-printing ink, fibroblasts (dermal layer cells) are spindle-shaped, while in the ink without laminin, the fibroblasts are hardly visible in the spindle shape. There was no significant difference in the epidermal cells in the two inks, which may be related to the intrinsic shape of the epidermal cells (in irregular circles).
Fig. 2 is a result of cell survival analysis, and it can be seen that as the diameter of the printing nozzle increases, the printing precision decreases, and after 7 days of printing, the cell still has significant survival ability, which indicates that the cell is not damaged by the pressing force during the printing process.
Example 3
This embodiment is different from embodiment 1 in that: the final concentrations of sodium alginate, gelatin, laminin and their cells in the ink were: 1% (w/v), 10% (w/v), 200. mu.g/ml and 5X 106/ml。
Example 4
This embodiment is different from embodiment 1 in that: the final concentrations of sodium alginate, gelatin, laminin and their cells in the ink were: 2% (w/v), 5% (w/v), 100. mu.g/ml and 5X 106/ml。
Example 5
The method for preparing the full-thickness skin model by using the dermal layer cell bioprinting ink in the embodiment 1 specifically comprises the following steps:
(1) adding the biological printing ink containing the dermal layer cells in the example 1 into a charging barrel of a 3D printer, mounting a printing needle and a piston, and then placing the printing needle and the piston into a precooled printer (10 ℃) to cool into gel, wherein the inner diameter of the needle is 0.33 mm;
(2) setting a dermis printing model, and extruding ink for printing, as shown in fig. 3 and 4, to form a dermis model; the dermis layer model comprises a first ink layer, a second ink layer, a third ink layer and a fourth ink layer, wherein the first ink layer, the second ink layer and the third ink layer are sequentially stacked, and the fourth ink layer is formed by printing along the peripheries of the first ink layer, the second ink layer and the third ink layer. In this embodiment, the top view of the model of the dermis layer is 16mm × 16 mm.
The first ink layer comprises two layers with transverse and longitudinal directions, and the line spacing of the first ink layer is 0 mm; the second ink layer comprises two layers which run transversely and longitudinally, and the line spacing between each layer in the second ink layer is 0.8 mm; the third ink layer comprises two layers which run transversely and longitudinally, the line spacing between each layer in the second ink layer is 1.6mm, the first ink layer and the fourth ink layer prevent epidermal cell suspension from seeping, the second ink layer and the third ink layer are arranged in a staggered mode, and a plurality of funnel-shaped spaces with gradually-increased openings are formed between the second ink layer and the third ink layer.
(3) The printed dermal layer model is placed into a calcium chloride aqueous solution with the mass concentration of 2% to be crosslinked into gel, and the gel is washed by normal saline, placed on a sterile stainless steel mesh bracket as shown in figure 5 and then placed in a sterile culture dish. Removing water from the surface of dermal tissue, spreading a 100 μ g/ml laminin solution on the upper surface, incubating at 37 deg.C for 1 hr, and spreading an epidermal cell suspension (100 μ l, 2 × 10)7HaCat cells/ml) is placed still in an incubator for 1 hour, then a proper amount of epidermal differentiation culture medium is added until the lower edge of an epidermal layer, the upper part of the epidermal layer is contacted with air, the lower part of the epidermal layer is contacted with the culture medium, and then gas-liquid phase culture is carried out, as shown in figure 6, complete epidermal and dermal layers are formed, and the printed skin structure is very close to the skin of a real human body.
Comparative example 3
This comparative example differs from example 1 in that: without the addition of laminin solution, the printing ink is not conducive to epidermal cell adhesion.
Comparative example 4
This comparative example differs from example 1 in that: GelMA ink is adopted, and the printing ink is not beneficial to the adhesion of epidermal cells.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A dermal bioprinting ink, comprising: comprises 50-200 mug/mL of laminin solution, wherein the laminin solution contains 3-10% of gelatin and 0.5-2% of sodium alginate by mass concentration.
2. Preparing a dermal bioprinting ink according to claim 1, wherein: the method comprises the following steps:
(1) placing sodium alginate powder and gelatin powder under ultraviolet lamp for irradiation, adding DMEM complete culture medium, dissolving, and sterilizing to obtain mixture of sodium alginate and gelatin;
(2) and (2) adding the mixture of sodium alginate and gelatin in the step (1) into the laminin solution to form the skin bioprinting ink.
3. The dermal bioprinting ink of claim 2, wherein: the cultured dermal cells were trypsinized, suspended in laminin solution, and mixed with sodium alginate and gelatin.
4. The dermal bioprinting ink of claim 2, wherein: the final concentration of the dermal layer cells is 5 × 106/ml。
5. A method of preparing a full-thickness skin model using the skin bioprinting ink of claim 1, wherein: the method comprises the following steps:
(1) placing sodium alginate powder and gelatin powder under ultraviolet lamp for irradiation, adding DMEM complete culture medium, dissolving, and sterilizing to obtain mixture of sodium alginate and gelatin;
(2) digesting the cultured dermal layer cells by trypsin, suspending the cells by a laminin solution, and then adding a mixture of sodium alginate and gelatin to form biological printing ink containing the dermal layer cells;
(3) after the dermal layer cell biological printing ink is cooled to gel, placing the gel in a charging barrel of a 3D printer, setting a dermal layer printing model, and extruding the ink for printing to form a dermal layer model;
the dermis layer model comprises a first ink layer, a second ink layer, a third ink layer and a fourth ink layer, wherein the first ink layer, the second ink layer and the third ink layer are sequentially laminated, the fourth ink layer encloses the first ink layer, the second ink layer and the third ink layer, and a funnel-shaped space with a plurality of gradually-increased openings is formed between the second ink layer and the third ink layer;
(4) and (2) crosslinking the printed dermis model into gel, cleaning, placing on a porous support, placing the support in a culture dish, paving a laminin solution on the upper surface of the third ink layer, placing in an incubator for incubation, paving a epithelial cell suspension, performing static culture in the incubator, adding an epidermal differentiation culture medium into the culture dish until the part below the epidermal cell suspension, and performing gas-liquid phase culture to form the full-layer skin model.
6. The method of preparing a full-thickness skin model with skin bioprinting ink according to claim 5, wherein: and (4) putting the dermis model into a calcium chloride aqueous solution with the mass concentration of 2% to crosslink into gel.
7. The method of preparing a full-thickness skin model with skin bioprinting ink according to claim 5, wherein: the first ink layer comprises two layers which are arranged in the transverse direction and the longitudinal direction, and the line spacing of the first ink layer is 0 mm.
8. The method of preparing a full-thickness skin model with skin bioprinting ink according to claim 7, wherein: the second ink layer comprises two layers running transversely and longitudinally, and the line spacing between each layer in the second ink layer is 0.8 mm.
9. The method of preparing a full-thickness skin model with skin bioprinting ink according to claim 8, wherein: the third ink layer comprises two layers running transversely and longitudinally, and the line spacing between each layer in the second ink layer is 1.6 mm.
10. The method of preparing a full-thickness skin model with skin bioprinting ink according to claim 5, wherein: the porous bracket is a stainless steel mesh or a plastic bracket.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116099056A (en) * | 2023-04-07 | 2023-05-12 | 云南云科特色植物提取实验室有限公司 | Artificial skin containing bionic texture structure and preparation method thereof |
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