Skin model manufacturing process based on cell sheet transfer printing technology and polypeptide-containing hydrogel

Abstract

The invention relates to a skin model manufacturing process based on a cell sheet transfer printing technology and polypeptide-containing hydrogel, belongs to the field of biological manufacturing, and comprises a cell sheet manufacturing and transfer printing technology and a polypeptide-containing hydrogel manufacturing technology. The manufacturing and transfer printing technology of the cell sheet comprises preparation of PDMS, hydrophilic modification of the surface of the PDMS, culture of the cell sheet and a transfer printing technology of the cell sheet, and simple and efficient manufacturing of the cell sheet can be achieved. According to the manufacturing technology of the polypeptide-containing hydrogel as the bionic basement membrane, the polypeptide is adopted to modify the hydrogel with mechanical strength, so that the adhesion of cells is improved, the hydrogel also has certain mechanical strength, and pores of the hydrogel are also beneficial to communication between the cells and exchange of nutrient substances. According to the skin model manufacturing process based on the cell sheet transfer printing technology and the polypeptide-containing hydrogel, the bionic property and efficiency of constructing an in-vitro skin tissue model are greatly improved, and progress and development of constructing the in-vitro tissue model can be promoted.

Description

Technical Field

[0001] The invention relates to the technical fields of biomanufacturing and tissue engineering, and in particular to a skin model manufacturing process based on cell membrane transfer technology and containing polypeptide hydrogel. Background Art

[0002] At present, the main methods used to make cell membrane sheets are temperature-sensitive culture systems, enzyme-sensitive culture systems, light-sensitive culture systems, ion-sensitive culture systems, and pH-sensitive culture systems. Among them, the temperature-sensitive culture system constructed by temperature-sensitive materials has been widely used in the preparation of tissue engineering cell sheets due to its advantages such as simple operation and easy control. Many temperature-sensitive materials have a lower critical solubility temperature (LCST). When the external temperature is higher than the LCST, it is hydrophobic, and cells can adhere to its surface and grow to form cell sheets. When the temperature is lower than the LCST, it gradually changes from hydrophobic to hydrophilic, which is not conducive to cell adhesion, resulting in natural cell detachment, and then a complete cell sheet structure can be obtained; by introducing enzyme-sensitive materials into the culture substrate, inoculating cells for culture, and after the cells proliferate to form a complete sheet structure, adding specific enzymes to degrade the substrate, it can also be used to obtain cell sheets. The advantage is that the substrate material has good biocompatibility, and the raw materials are easy to obtain and low cost; based on light-sensitive materials, a light-responsive culture system is constructed, and the adhesion of the substrate to cells is changed by light, which can also be used to prepare and harvest cell sheets. This method is simple to operate and causes little direct damage to cells. The ion-sensitive culture system generally utilizes the response characteristics of the substrate material to ions. When the ion content changes, the substrate material will be degraded or dissociated, thereby releasing the cells growing on the surface with almost no damage to the cells. The pH-sensitive system is mainly based on pH-responsive materials. By changing the environmental pH value, the culture substrate material is degraded or dissociated, causing the cells adhering to its surface to naturally detach and obtain cell sheets.

[0003] The materials currently used to make biomimetic basement membranes include simple porous polymer membranes, highly structured electrospun fibers, and 3D hydrogel systems. Although porous polymer membranes are low-cost, easy to handle, and durable, their mechanical and chemical properties are static and not fully suitable for replacing and mimicking the complexity of BM. Electrospinning is a versatile technology that can produce micro- to nanofiber scaffolds that can be modulated based on their surface morphology, fiber diameter size, mesh thickness, and fiber arrangement. The potential to achieve desired mechanical and chemical properties by changing electrospinning parameters has been developed for customized BM mimics.

[0004] However, electrospinning is limited in terms of reproducibility due to its high dependence on environmental factors such as humidity and temperature. The choice of solvents and high pressure used during electrospinning can also lead to the loss of bioactivity of biomolecules. In a way, synthetic hydrogels are similar to ECMs in that there is a polymer network formed through covalent and non-covalent interactions in a water-swollen environment. Fine-tuning their bulk mechanical properties by controlling cross-linking and molecular density facilitates their use as BM mimics. Biofunctionalization of hydrogels is achieved by incorporating short peptide sequences found in proteins such as collagen and laminin, which enhance cell binding to hydrogel mimics through integrins. Another aspect is the ability to artificially enrich synthetic hydrogels with growth factors that regulate various cellular interactions and behaviors similar to the natural BM microenvironment. 3D hydrogels containing cells can be used to mimic complex tissue structures using printing techniques such as microextrusion, laser-assisted, inkjet, and stereolithography, and their outstanding properties show great promise in a wide range of biomedical applications. Summary of the invention

[0005] 1. Technical issues to be resolved In view of the shortcomings of the prior art, the purpose of the present invention is to develop a skin model manufacturing process based on cell membrane manufacturing, transfer and polypeptide hydrogel, so as to improve the bionics and efficiency of constructing in vitro skin tissue models and promote the progress and development of constructing in vitro tissue models.

[0006] (II) Technical solution To achieve the above-mentioned purpose, the present invention provides the following technical solutions: a novel cell membrane manufacturing and transfer process, including a cell membrane manufacturing and transfer technology and a polypeptide-containing hydrogel manufacturing technology, wherein the required cell membrane is manufactured through simple operation and can be quickly used for transfer, and a hydrogel with mechanical strength is modified by polypeptide to improve the adhesion of cells while also having a certain mechanical strength, and the pores of the hydrogel are also conducive to communication between cells and the exchange of nutrients.

[0007] Preferably, the manufacturing and transfer process of the cell membrane sheet includes the preparation of PDMS, hydrophilic modification of the PDMS surface, cultivation of the cell membrane sheet and transfer technology of the cell membrane sheet. The preparation of PDMS is obtained by mixing the purchased prepolymer and the curing agent in a ratio of 10:1 and then curing; the hydrophilic modification of PDMS is achieved by first plasma treatment and then soaking in PVA solution; the cultivation of the cell membrane sheet is achieved by first sterilizing the modified PDMS with ultraviolet light, then soaking it in PBS to remove alcohol, and then inoculating cells on the PDMS until the cells are fully grown; the transfer technology of the cell membrane sheet is achieved by placing the cell membrane sheet on a special transfer surface moistened with culture medium and then waiting for a period of time.

[0008] Preferably, the manufacturing process of the polypeptide-containing hydrogel as a biomimetic basement membrane is as follows: the selected polypeptide is first dissolved in a HEPES buffer solution, PEGDA polyethylene glycol diacrylate with a molecular weight of 600 is added and stirred to dissolve, and finally a photoinitiator LAP phenyl-2,4,6-trimethylbenzoylphosphinate lithium is added and quickly stirred to dissolve, and the obtained solution is irradiated with an ultraviolet lamp for photocuring to obtain a polypeptide-modified hydrogel.

[0009] A manufacturing process for a hydrogel containing polypeptides as a bionic basement membrane uses polypeptides to modify a hydrogel with mechanical strength, thereby improving cell adhesion while also having a certain mechanical strength. The pores of the hydrogel are also conducive to communication between cells and exchange of nutrients.

[0010] The manufacturing process of the polypeptide-containing hydrogel as a biomimetic basement membrane comprises the steps of first dissolving the selected polypeptide in a HEPES buffer, then adding PEGDA (polyethylene glycol diacrylate) with a molecular weight of 600 and stirring to dissolve, and finally adding a photoinitiator LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate) and rapidly stirring to dissolve, and irradiating the obtained solution with an ultraviolet lamp for photocuring to obtain a polypeptide-modified hydrogel.

[0011] A method for manufacturing a skin model based on cell membrane fabrication, transfer and polypeptide hydrogel, comprising the following steps: a. Place the hydrogel containing the polypeptide under ultraviolet light for 30 minutes for sterilization; b. Remove all the culture medium on the surface of the cultured cell membrane, then rinse along the side wall of the culture dish with PBS, and then wet the surface of the hydrogel with the culture medium; c. Use tweezers to slowly pick up the cell membrane sheet, and then slowly place it on the surface of the hydrogel, and then place both together in an incubator at 37°C and 5% CO2 for a period of time; d. Take the hydrogel out of the incubator, remove the PDMS with tweezers, add culture medium, and then place the hydrogel in the incubator for culture; e. Change the medium in time until the cell membrane forms a stable connection with the hydrogel basement membrane, and then change the culture medium to differentiate the cells; f. After culturing for a period of time, the skin expression products are measured to verify the biological function of the obtained skin model.

[0012] (III) Beneficial effects Compared with the prior art, the present invention provides a skin model manufacturing process based on cell membrane transfer technology and polypeptide hydrogel, which has the following beneficial effects: This skin model manufacturing process based on cell membrane transfer technology and polypeptide hydrogel adopts a new cell membrane manufacturing process, the materials are simple and easy to obtain, the cost is low, and the efficiency is high. The new process is used to manufacture the hydrogel bionic basement membrane, and polypeptide modification is used to improve cell adhesion. At the same time, it has good mechanical properties and biological properties; This design combines the above two processes, which is beneficial to improve the bionics and efficiency of constructing an in vitro skin tissue model, and the prepared skin model has good biological properties; the process described in the present invention utilizes cell membrane manufacturing, transfer technology and polypeptide hydrogel manufacturing technology to achieve efficient and bionic manufacturing of in vitro skin tissue models; the process has the advantages of simple operation, easy availability of materials, strong reproducibility, mass production, good biological functionality, etc., and is suitable for constructing an in vitro skin tissue model. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The schematic diagram of the process of manufacturing PDMS provided by the present invention.

[0014] Figure 2 Schematic diagram of the process for fabrication of the novel cell membrane sheet.

[0015] Figure 3 Schematic diagram of the process for fabricating hydrogels containing peptides.

[0016] Figure 4 Schematic diagram of the cell transfer technique.

[0017] Figure 5 It is a simplified process flow chart of the entire invention.

[0018] In the figure: 1. weighing balance; 2. prepolymer; 3. curing agent; 4. vacuum machine; 5. oven; 6. PDMS; 7. PVA solution; 8. heating stirrer; 9. plasma processor; 10. UV lamp; 11. PEGDA; 12. HEPES; 13. polypeptide; 14. LAP; 15. magnetic stirrer; 16. polypeptide-containing hydrogel; 17. cell membrane. DETAILED DESCRIPTION

[0019] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Embodiment 1: A skin model manufacturing process based on cell membrane transfer technology and hydrogel containing polypeptide 13, including the manufacturing and transfer technology of cell membrane and the manufacturing technology of hydrogel containing polypeptide 13. The required cell membrane is manufactured through simple operation and can be quickly used for transfer. A hydrogel with mechanical strength is modified by polypeptide 13 to improve the adhesion of cells while also having a certain mechanical strength. The pores of the hydrogel are also conducive to communication between cells and the exchange of nutrients.

[0021] The manufacturing and transfer process of the cell membrane sheet includes the preparation of PDMS-6, the hydrophilic modification of the surface of PDMS-6, the culture of the cell membrane sheet and the transfer technology of the cell membrane sheet; the preparation of PDMS-6 is obtained by mixing the purchased prepolymer 2 and the curing agent 3 in a ratio of 10:1 and then curing; the hydrophilic modification of PDMS-6 is achieved by first plasma treatment and then soaking in PVA solution 7; the culture of the cell membrane sheet is achieved by first sterilizing the modified PDMS-6 with ultraviolet light, then soaking it in PBS to remove alcohol, and then inoculating cells on the PDMS-6 to wait for the cells to grow; the transfer technology of the cell membrane sheet is achieved by placing the cell membrane sheet on a special transfer surface moistened with a culture medium and then waiting for a period of time.

[0022] The invention discloses a manufacturing process of a hydrogel containing polypeptide 13 as a biomimetic basement membrane, wherein the selected polypeptide 13 is first dissolved in a HEPES buffer solution, and then PEGDA-11 polyethylene glycol diacrylate with a molecular weight of 600 is added and stirred to dissolve, and finally a photoinitiator LAP phenyl-2,4,6-trimethylbenzoyl phosphinate lithium is added and quickly stirred to dissolve, and the obtained solution is irradiated with an ultraviolet lamp 10 for photocuring, thereby obtaining a hydrogel modified with polypeptide 13.

[0023] The skin model manufacturing process based on cell membrane transfer technology and peptide 13 hydrogel includes the following steps: 1) Manufacturing PDMS-6: Use a weighing balance 1 to weigh prepolymer 2 and curing agent 3. Stir PDMS-6 evenly at a ratio of prepolymer 2: curing agent 3 = 10:1 and pour it into a culture dish. Then put it into a vacuum machine and wait for 20 minutes to eliminate bubbles. Then place it in a 60°C oven 5 for four hours to dry and cure. 2) Modify the hydrophilicity of PDMS-6: first, place the cured PDMS-6 in a plasma treatment machine 99 and treat it with ultraviolet light for 1 minute and 40 seconds to preliminarily change the hydrophilicity of the PDMS-6 surface. At the same time, add PVA polyvinyl alcohol into deionized water, stir and dissolve it at a high temperature of 70°C for 30 minutes to prepare a 1% concentration of PVA solution 7, then pour the prepared solution on the surface of the plasma-treated PDMS-6 for permanent modification, then let it stand for 30 minutes, pour out the solution and rinse with deionized water; place the rinsed PDMS-6 in a 60°C oven 5 to dry for 1 hour, and finally sterilize it with ultraviolet light for 30 minutes; 3) Preparation of cell membrane: Epidermal cells are inoculated on the modified PDMS-6 surface, and then placed in an incubator at 37°C and 5% CO2 for 2 days until the cells grow all over the PDMS-6 surface to obtain a cell membrane; 4) Preparation of hydrogel containing polypeptide 13: Firstly, the selected polypeptide 13 was dissolved in HEPES buffer for 30 minutes, then PEGDA-11 polyethylene glycol diacrylate with a molecular weight of 600 was added and allowed to stand at room temperature for 1 hour, and finally a photoinitiator LAP phenyl-2,4,6-trimethylbenzoylphosphinate lithium was added and rapidly stirred for 30 minutes to dissolve, and the obtained solution was irradiated with a UV lamp for 10 minutes for photocuring to obtain a hydrogel modified with polypeptide 13; 5) Transferring the cell membrane: First, place the hydrogel containing peptide 13 under ultraviolet light for 30 minutes for sterilization, then remove all the culture medium on the surface of the cultured cell membrane, then rinse along the side wall of the culture dish with PBS, and then moisten the surface of the hydrogel with the culture medium; then slowly clamp the cell membrane with tweezers and slowly place it on the surface of the hydrogel, and finally place both together in a 37°C, 5% CO2 incubator for a period of time to complete the transfer; 6) Post-culture of the in vitro skin tissue model: first take the hydrogel out of the incubator, remove the PDMS-6 with tweezers, then add culture medium, and then place the hydrogel in the incubator for culture; change the medium in time until the cell membrane forms a stable connection with the hydrogel basement membrane, and then replace the culture medium for cell differentiation. After a period of culture, measure the skin expression to verify the biological function and differentiation of the obtained skin model.

[0024] The beneficial effects of the present invention are: the skin model manufacturing process based on cell membrane transfer technology and polypeptide 13 hydrogel, by adopting a new cell membrane manufacturing process, has simple and easy-to-obtain materials, low cost and high efficiency, adopts a new process to manufacture the hydrogel bionic basement membrane, and uses polypeptide 13 modification to improve cell adhesion, while having good mechanical properties and biological properties; This design combines the above two processes, which is beneficial to improving the bionics and efficiency of constructing an in vitro skin tissue model, and the prepared skin model has good biological properties; The process of the present invention utilizes cell membrane manufacturing, transfer technology and polypeptide 13 hydrogel manufacturing technology to achieve efficient and bionic manufacturing of in vitro skin tissue models; The process has the advantages of simple operation, easy availability of materials, strong reproducibility, mass production, good biological functionality, etc., and is suitable for constructing an in vitro skin tissue model.

[0025] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A skin model manufacturing process based on cell membrane transfer technology and polypeptide hydrogel, including the manufacturing and transfer technology of cell membrane (17) and the manufacturing technology of hydrogel containing polypeptide (13), characterized in that: The required cell membrane sheets (17) can be manufactured through simple operations and can be quickly used for transfer.

2. The skin model manufacturing process based on cell membrane transfer technology and polypeptide hydrogel according to claim 1, characterized in that: The manufacturing and transfer process of the cell membrane sheet (17) includes the preparation of PDMS (6), hydrophilic modification of the surface of PDMS (6), cultivation of the cell membrane sheet (17) and transfer technology of the cell membrane sheet (17); the preparation of PDMS (6) is obtained by mixing the purchased prepolymer (2) and the curing agent (3) in a ratio of 10:1 and then curing; the hydrophilic modification of PDMS (6) is achieved by first plasma treatment and then soaking in PVA solution (7); the cultivation of the cell membrane sheet (17) is achieved by first sterilizing the modified PDMS (6) with ultraviolet light, then soaking in PBS to remove alcohol, and then inoculating cells on the PDMS (6) to obtain the cell membrane sheet; the transfer technology of the cell membrane sheet (17) is achieved by placing the cell membrane sheet (17) on a special transfer surface moistened with a culture medium and then waiting for a period of time.

3. The skin model manufacturing process based on cell membrane transfer technology and polypeptide hydrogel according to claim 1, characterized in that: The manufacturing process of the hydrogel containing the polypeptide (13) as a biomimetic basement membrane comprises the steps of first dissolving the selected polypeptide (13) in a HEPES (12) buffer solution, then adding PEGDA (11) polyethylene glycol diacrylate with a molecular weight of 600 and stirring to dissolve, and finally adding a photoinitiator LAP (14) phenyl-2,4,6-trimethylbenzoylphosphinate lithium and rapidly stirring to dissolve, and irradiating the obtained solution with an ultraviolet lamp (10) for photocuring to obtain a hydrogel modified with the polypeptide (13).

4. The skin model manufacturing process based on cell membrane transfer technology and polypeptide hydrogel according to claim 1, characterized in that: The steps include: 1) Manufacturing PDMS (6): Use a weighing balance (1) to weigh the prepolymer (2) and the curing agent (3). First, mix the PDMS (6) in a ratio of prepolymer (2): curing agent (3) = 10:1 and pour it into a culture dish. Then, put it into a vacuum machine and wait for 20 minutes to eliminate bubbles. Then, place it in a 60°C oven (5) for four hours to dry and cure. 2) Modifying the hydrophilicity of PDMS (6): first, placing the cured PDMS (6) in a plasma treatment machine (9) (9) and treating it with ultraviolet light for 1 minute and 40 seconds to preliminarily change the hydrophilicity of the surface of PDMS (6); at the same time, adding PVA polyvinyl alcohol into deionized water, stirring and dissolving it at a high temperature of 70°C for 30 minutes, and preparing a 1% concentration of PVA solution (7); then pouring the prepared solution onto the surface of the plasma-treated PDMS (6) for permanent modification; then, after standing for 30 minutes, pouring out the solution and rinsing it with deionized water; placing the rinsed PDMS (6) in a 60°C oven (5) for 1 hour, and finally sterilizing it by irradiating it with ultraviolet light for 30 minutes; 3) Preparation of a cell membrane sheet (17): Epidermal cells are inoculated on the surface of the modified PDMS (6), and then placed in an incubator at 37°C and 5% CO2 for 2 days until the cells grow all over the surface of the PDMS (6) to obtain a cell membrane sheet (17); 4) preparing a hydrogel containing a polypeptide (13): first dissolving the selected polypeptide (13) in a HEPES (12) buffer for 30 minutes, then adding PEGDA (11) polyethylene glycol diacrylate with a molecular weight of 600 and standing at room temperature for 1 hour, and finally adding a photoinitiator LAP (14) phenyl-2,4,6-trimethylbenzoylphosphinate lithium, stirring rapidly for 30 minutes to dissolve, and irradiating the resulting solution with an ultraviolet lamp (10) for 10 minutes for photocuring to obtain a hydrogel modified with the polypeptide (13); 5) Transferring the cell membrane (17): first place the hydrogel containing the polypeptide (13) under a UV lamp (10) for 30 minutes for sterilization, then remove all the culture medium on the surface of the cultured cell membrane (17), then rinse along the side wall of the culture dish with PBS, and then moisten the surface of the hydrogel with the culture medium; then slowly clamp the cell membrane (17) with tweezers and slowly place it on the surface of the hydrogel, and finally place both together in a 37°C, 5% CO2 incubator for a period of time to complete the transfer; 6) Post-cultivation of the in vitro skin tissue model: first, take the hydrogel out of the incubator, remove the PDMS (6) with tweezers, then add culture medium, and then place the hydrogel in the incubator for cultivation; change the medium in a timely manner until the cell membrane (17) forms a stable connection with the hydrogel basement membrane, and then replace the culture medium for cell differentiation. After a period of culture, measure the skin expression to verify the biological function and differentiation of the obtained skin model.

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