CN114540275B - Skin biological printing ink and preparation method and application thereof - Google Patents
Skin biological printing ink and preparation method and application thereof Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/52—Fibronectin; Laminin
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/74—Alginate
Abstract
The invention discloses a skin biological printing ink, which relates to the technical field of skin tissue engineering and comprises 50-200 mug/mL of 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 gelatin with the mass concentration of 3-10% and sodium alginate with the mass concentration of 0.5-2% to form the skin biological printing ink, which is favorable for proliferation of dermis cells, has high dermis cell survival rate, is favorable for growth and differentiation of epidermis cells when full-layer skin is further formed in the later stage, so that a compact and thicker epidermis is formed, and the dermis are connected in a staggered manner by arranging the dermis printing model, so that the connection part of the epidermis and the dermis is favorable for forming a wavy form, and the morphological structure of real skin is simulated.
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 a vital role in protecting the human body from the external environment. Since skin is the first line of defense for the human body, it is susceptible to different types of injury (e.g., traumatic wounds, burns, diabetic feet, etc.). When the skin is severely damaged and cannot repair itself, the current main treatment is autograft or tissue engineering skin adjuvant therapy. Because of the limited number of autologous skin grafts, it is particularly important to develop tissue engineering skin substitutes for the treatment of skin lesions. Furthermore, european legislation prohibits the use of animals for testing cosmetic ingredients and increases the number of industrial chemicals that must be risk assessed. Thus, there is also a strong need in the cosmetic and pharmacological fields for reliable, high quality, repeatable skin substitutes to replace animals for testing.
Bioprinting technology has been vigorously developed in the field of biological manufacturing, which can manipulate the spatial distribution of cells and extracellular matrix, simulating the biological structure of tissues or organs. The rapid development of bioprinting technology provides a new idea for tissue engineering skin construction, and some researchers have demonstrated the potential of bioprinting skin tissues, wherein the printed skin tissues gradually develop from simple to complex, low biomimetic performance to high biomimetic performance. Lee et al were first printing skin tissue using bioprinting techniques based on a Multi-layer construction method (collagen-fibroblast-collagen-keratinocyte-collagen) (Lee, w., et al, multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrics, 2009.30 (8): p.1587-95.); binder et al first used a pig wound model of back skin defects for in vivo bioprinting, and based on inkjet bioprinting techniques, cell-containing bio-ink was directly printed onto the damaged skin surface, showing that the epithelialization rate of the printed group was significantly higher than that of the control group (Binder KW, zhao W, aboushrub T, et al in situ bioprinting of the skin for burns.J Am Coll Surg 2011; 211:S76.). Although some progress has been made in skin bioprinting, currently printed skin is not widely used, mainly because the current skin printing technology is relatively complex to operate and is not suitable for mass preparation of skin samples, and in addition, the current printed skin samples are not high in imitation and reduce the applicability.
A key factor in 3D printing of skin is the raw material for 3D bio-printing, i.e. bio-ink. The in vitro skin model or skin disease model can be used for researching the mechanism of skin diseases, skin irritation test, anti-aging compound test and the like. However, at present, the construction of a personalized and customized full-layer 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 to operate, has high cost, is difficult to prepare in batches, is difficult to form a compact epidermis layer, and cannot be well applied to subsequent researches. The patent application publication No. CN109054503A discloses a preparation method, a printing method and application of the bio-printing ink, but the bio-printing ink is not suitable for skin bio-printing.
Disclosure of Invention
One of the technical problems to be solved by the invention is to provide a biological printing ink suitable for skin.
The invention solves the technical problems by the following technical means:
a skin biological printing ink comprises 50-200 mug/mL of 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 beneficial effects are that: the laminin solution contains gelatin with the mass concentration of 3-10% and sodium alginate with the mass concentration of 0.5-2% to form the skin bioprinting ink, which is favorable for proliferation of dermis cells, has high survival rate of dermis cells, and is favorable for growth and differentiation of epidermis cells when full-layer skin is formed in the later stage, so that a compact and thicker epidermis 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 sodium alginate and gelatin mixture;
(2) And (3) adding the sodium alginate and gelatin mixture obtained in the step (1) into the laminin solution to form the skin bioprinting ink.
The beneficial effects are that: the inventive layer adds gelatin with mass concentration of 3-10% and sodium alginate with mass concentration of 0.5-2% into the fibronectin solution to form the skin biological printing ink, which is beneficial to proliferation of dermis layer cells, has high cell survival rate, and is beneficial to growth and differentiation of epidermis layer cells when forming full-layer skin in later period, thereby forming compact and thicker epidermis layer.
Preferably, the cultured dermal cells are digested with trypsin, the cells are suspended with a laminin solution, and then a mixture of sodium alginate and gelatin is added.
Preferably, the final concentration of cells in the dermis layer is 5X 10 6 /ml。
The second technical problem to be solved by the invention is that the existing skin printing biological ink is unfavorable for the attachment of the epidermis layer, so that a complete and compact epidermis layer cannot be formed, and a method for preparing a full-layer skin model by adopting the skin biological printing ink is provided.
A method for preparing a full-thickness skin model using skin bioprinting ink, comprising the steps of:
(1) Placing sodium alginate powder and gelatin powder under ultraviolet lamp for irradiation, adding DMEM complete culture medium, dissolving, and sterilizing to obtain sodium alginate and gelatin mixture;
(2) Digesting the cultured dermis layer cells with trypsin, suspending the cells with laminin solution, and then adding a mixture of sodium alginate and gelatin to form the bioprinting ink containing the dermis layer cells;
(3) After the dermis layer cell biological printing ink is cooled to be gel, placing the dermis layer cell biological printing ink in a material cylinder of a 3D printer, setting a dermis layer printing model, extruding the ink for printing, and forming a dermis 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 plurality of funnel-shaped spaces with gradually increased openings are formed between the second ink layer and the third ink layer;
(4) And (3) crosslinking the printed dermis layer model into gel, cleaning, placing the gel on a porous bracket, placing the bracket in a culture dish, paving a laminin solution on the upper surface of a third ink layer, placing the gel in an incubator for incubation, paving an epidermis cell suspension, standing in the incubator for culture, adding an epidermis differentiation culture medium into the culture dish until the epidermis cell suspension is below, and performing gas-liquid phase culture to form the full-layer skin model.
The beneficial effects are 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 bioprinting ink, so that proliferation of dermis cells is facilitated, the cell survival rate is very high, attachment of epidermis is facilitated, a complete and compact epidermis is formed, the dermis and the dermis are connected in a staggered manner by arranging a dermis printing model, the connection part of the epidermis and the dermis is facilitated to form a wavy shape, and the morphological structure of real skin can be simulated.
The dermis layer cell biological printing ink can be extruded after being cooled to be gel, and can not be printed and extruded in a drop shape if the cooling ink is not cooled to be liquid.
The traditional gas-liquid phase culture uses a commercial transwell, however, the cost of the vessel is very high, the capacity of the storage culture medium is very limited, so that the culture medium needs to be replaced frequently in the culture process, and the precious energy of scientific researchers is occupied.
Preferably, in the step (4), the dermis layer model is put into a calcium chloride aqueous solution with the mass concentration of 2% to crosslink into gel.
Preferably, the first ink layer comprises two layers which are transversely and longitudinally oriented, and the line spacing of the first ink layer is 0mm.
Preferably, the second ink layer comprises two layers running in the transverse and longitudinal directions, the line spacing between each layer in the second ink layer being 0.8mm.
Preferably, the third ink layer comprises two layers running in the transverse and longitudinal directions, the line spacing between each layer in the second ink layer being 1.6mm.
Preferably, the porous scaffold is a stainless steel mesh or a plastic scaffold.
The beneficial effects are that: the stainless steel net or the plastic bracket has very low cost and can be repeatedly used after disinfection.
Preferably, the concentration of the laminin solution in step (3) is 100 μg/mL.
Preferably, the concentration of the epithelial cell suspension in step (3) is 2X 10 7 /ml。
The invention has the advantages that: the laminin solution contains gelatin with the mass concentration of 3-10% and sodium alginate with the mass concentration of 0.5-2% to form the skin bioprinting ink, which is favorable for proliferation of dermis cells, has high survival rate of dermis cells, and is favorable for growth and differentiation of epidermis cells when full-layer skin is formed in the later stage, so that a compact and thicker epidermis 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 bioprinting ink, so that proliferation of dermis cells is facilitated, the cell survival rate is very high, attachment of epidermis is facilitated, a complete and compact epidermis is formed, the dermis and the dermis are connected in a staggered manner by arranging a dermis printing model, the connection part of the epidermis and the dermis is facilitated to form a wavy shape, and the morphological structure of real skin can be simulated.
The traditional gas-liquid phase culture uses a commercial transwell, however, the cost of the vessel is very high, the capacity of the storage culture medium is very limited, so that the culture medium needs to be replaced frequently in the culture process, and the precious energy of scientific researchers is occupied.
The stainless steel net support has very low cost and can be repeatedly used after disinfection.
Drawings
FIG. 1 is a schematic diagram of the cells of example 1, example 2 and comparative example 1 of the present invention in an ink; in the figure, A is the epidermal cells obtained by digestion in example 2, B is the epidermal cells in example 2 grown in the ink, C is the epidermal cells in comparative example 2 grown in the ink without laminin, D is the dermal cells obtained by digestion in example 1, E is the dermal cells in example 1 grown in the ink, and F is the dermal cells in comparative example 1 grown in the ink without laminin;
FIG. 2 is a graph of cell survival analysis according to the present invention; the figures A-C show the shapes (0.33 mm,0.41mm,0.51 mm) printed with nozzles of no size; D-F represents the corresponding cell survival analysis after 7 days of culture;
FIG. 3 is a schematic diagram of a dermis layer model in example 5 of the present invention; the first ink layer is shown in the figure, the second ink layer is shown in the figure, and part of the fourth ink layer is shown in the figure, and the dermis layer is shown in the figure;
FIG. 4 is a diagram showing the real leather layer model printing process in example 5 of the present invention;
FIG. 5 is a diagram showing the printing process and the air-liquid culture of the dermis layer model in example 5 of the present invention;
FIG. 6 is a graph showing the results of HE staining of full-thickness skin obtained in example 5 of the present invention; in the graph, A is the HE staining result of human skin tissue, and B-D is the HE staining result of printing skin tissue cultured for different days.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The test materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Those of skill in the art, without any particular mention of the techniques or conditions, may follow the techniques or conditions described in the literature in this field or follow the product specifications.
Example 1
The preparation of the bioprinting ink containing the dermis layer cells comprises the following steps:
(1) Placing sodium alginate powder and gelatin powder under ultraviolet lamp for irradiation for 30min, adding 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 into a 60 ℃ oven for 1h to fully dissolve sodium alginate and gelatin, placing the mixture of sodium alginate and gelatin into the 60 ℃ oven for pasteurization for 1h after the mixture of sodium alginate and gelatin is cooled to room temperature to obtain the mixture of sodium alginate and gelatin, and placing the mixture into a cell incubator for standby;
(2) Cell culture: firstly, sucking out the original culture medium in a culture dish by a pipetting gun; then, washing with PBS for 3 times, removing residual culture medium, adding 2mL pancreatin (0.25% Trypsin-EDTA), putting back into an incubator for digestion, and adding 2mL DMEM complete culture medium to stop digestion when the cells become round and some cells float for about 3 min; finally, the cells were gently scraped off the bottom of the dish, the liquid was then transferred to a centrifuge tube, centrifuged (120 g,5 min) in the centrifuge, the supernatant was discarded, and fresh medium was added for resuspension.
(2) Cell resuspension was performed with 100 μg/ml of laminin solution (solvent physiological saline), and then the laminin-containing cell suspension was mixed with sodium alginate-gelatin mixture 1:1 to form the final bioprinting ink containing dermis layer cells, wherein the final concentration of sodium alginate, gelatin, laminin and cells thereof is as follows: 1% (w/v), 5% (w/v), 50. Mu.g/ml and 5X 10) 6 /ml。
Example 2
This embodiment differs from embodiment 1 in that: the dermis layer cells are replaced with the epidermis cells.
Comparative example 1
This embodiment differs from embodiment 1 in that: the laminin solution was replaced with an equal amount of physiological saline.
Comparative example 2
This embodiment differs from embodiment 1 in that: the dermis layer cells were replaced with epidermis cells, while the laminin solution was replaced with an equal amount of physiological saline.
FIG. 1 shows the morphology of cells in a bioprinting ink, and it can be seen that fibroblasts (dermis layer cells) are spindle-shaped when laminin is contained in the bioprinting ink, whereas in laminin-free ink, almost no spindle-shaped fibroblasts are seen. There is no significant difference in the epidermal cells in the two inks, which may be related to the shape of the epidermal cells themselves (in irregular circles).
Fig. 2 is a graph showing the results of cell survival analysis, and it can be seen that the printing accuracy decreases with increasing diameter of the printing nozzle, and the cells still have significant viability after 7 days of printing, indicating that the extrusion force during printing does not damage the cells.
Example 3
This embodiment differs from embodiment 1 in that: the final concentration of sodium alginate, gelatin, laminin and cells in the ink is: 1% (w/v), 10% (w/v), 200. Mu.g/ml and 5X 10 6 /ml。
Example 4
This embodiment differs from embodiment 1 in that: the final concentration of sodium alginate, gelatin, laminin and cells in the ink is: 2% (w/v), 5% (w/v), 100. Mu.g/ml and 5X 10) 6 /ml。
Example 5
The method for preparing the full-layer skin model by adopting the dermal layer cell bioprinting ink in the embodiment 1 specifically comprises the following steps:
(1) The biological printing ink containing dermal cells in the embodiment 1 is added into a 3D printer charging barrel, a printing needle head and a piston are arranged, and then the biological printing ink is cooled into gel in a precooled printer (10 ℃), and the inner diameter of the needle head is 0.33mm;
(2) Setting a dermis layer printing model, extruding ink for printing, and forming a dermis layer model as shown in fig. 3 and 4; 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, 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. The planar size of the dermis layer model in this example is 16mm by 16mm in plan view.
The first ink layer comprises two layers which are transversely and longitudinally arranged, and the line spacing of the first ink layer is 0mm; the second ink layer comprises two layers which are transversely and longitudinally oriented, and the line spacing between each layer in the second ink layer is 0.8mm; the third ink layer comprises two layers which transversely run and longitudinally run, the line interval between each layer in the second ink layer is 1.6mm, the first ink layer and the fourth ink layer prevent the epidermal cell suspension from being exosmosed, 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) Will print the dermis layerThe model was put into a 2% calcium chloride aqueous solution to crosslink into gel, washed with physiological saline, and then put on a sterile stainless steel mesh support as shown in fig. 5, and then put in a sterile petri dish. Removing water from the surface of dermal tissue, spreading a layer of laminin solution (100. Mu.g/ml) on the surface, incubating in a 37℃incubator for 1 hr, and spreading a layer of epidermal cell suspension (100. Mu.l, 2X 10) 7 After resting in an incubator for 1h,/ml HaCat cells), a suitable amount of epidermal differentiation medium was added until the lower edge of the epidermis, above which was in contact with air, and below which was in contact with the medium, so as to perform gas-liquid culture, as shown in fig. 6, to form complete epidermis and dermis layers, the printed skin structure being very close to that 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 unfavorable for the attachment of epidermal cells.
Comparative example 4
This comparative example differs from example 1 in that: the GelMA ink is adopted, and the printing ink is unfavorable for the attachment of epidermal cells.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (6)
1. The method for preparing the full-layer skin model by adopting the skin biological printing ink is characterized by comprising the following steps of: 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 sodium alginate and gelatin mixture;
(2) The cultured dermis layerCells are digested by trypsin, cells are suspended by laminin solution, and then a mixture of sodium alginate and gelatin is added to form the bioprinting ink containing dermal cells, wherein the final concentration of the sodium alginate, gelatin, laminin and dermal cells in the bioprinting ink is as follows: 1% (w/v), 5% (w/v), 50. Mu.g/ml and 5X 10) 6 /ml；
(3) Cooling and gelling the cell biological printing ink containing the dermis layer, then placing the cell biological printing ink into a material cylinder of a 3D printer, setting a dermis layer printing model, extruding the ink for printing, and forming the dermis 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 plurality of funnel-shaped spaces with gradually increased openings are formed between the second ink layer and the third ink layer;
(4) Crosslinking the printed dermis layer model into gel, cleaning, placing on a porous bracket, placing the bracket in a culture dish, spreading laminin solution with 100 μg/ml on the upper surface of a third ink layer, placing in an incubator for incubation, and spreading laminin solution with concentration of 2×10 7 After the culture of the epidermal cell suspension in an incubator by standing, an epidermal differentiation medium is added into a culture dish until the epidermal cell suspension is below, and gas-liquid phase culture is carried out to form a full-layer skin model.
2. The method for preparing a full-thickness skin model from the skin bioprinting ink according to claim 1, wherein: and (4) putting the dermis layer model into a calcium chloride aqueous solution with the mass concentration of 2% to crosslink into gel.
3. The method for preparing a full-thickness skin model from the skin bioprinting ink according to claim 1, wherein: the first ink layer comprises two layers which are transversely and longitudinally arranged, and the line spacing of the first ink layer is 0mm.
4. The method for preparing a full-thickness skin model from the skin bioprinting ink according to claim 1, wherein: 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 0.8mm.
5. The method for preparing a full-thickness skin model from the skin bioprinting ink according to claim 1, wherein: the third ink layer comprises two layers which run transversely and longitudinally, and the line spacing between each layer in the third ink layer is 1.6mm.
6. The method for preparing a full-thickness skin model from the skin bioprinting ink according to claim 1, wherein: the porous bracket is a stainless steel net or a plastic bracket.
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