A 3D printed scaffold for large-scale culture of umbilical cord mesenchymal stem cells

Abstract

The present invention belongs to the technical field of cell culture materials, and specifically relates to a 3D printed scaffold for large-scale culture of umbilical cord mesenchymal stem cells. The above-mentioned 3D printed scaffold for large-scale culture of umbilical cord mesenchymal stem cells includes a scaffold body, the scaffold body is a 3D printed integrated structure, cell culture wells are arranged on the scaffold body, and the cell culture wells are filled with a composite gel loaded with umbilical cord mesenchymal stem cells. The pore structure on the scaffold can optimize cell attachment, proliferation and nutrient exchange; the scaffold body and the composite gel are used in combination to have good compatibility and wall adhesion rate of umbilical cord mesenchymal stem cells, and the composite gel has the characteristic of being able to dissociate quickly under specific stimulation, so that cells can be collected safely and efficiently without damaging the cell state.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture materials, and in particular to a 3D printed scaffold for large-scale culture of umbilical cord mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs) have immunomodulatory functions, are easy to separate, culture, expand and purify, and still have stem cell characteristics after multiple passages and expansions, and have strong proliferation ability and multidirectional differentiation potential. Among them, umbilical cord mesenchymal stem cells (UCMSCs) derived from the placenta have the advantages of abundant sources, non-invasive acquisition, low immunogenicity, strong in vitro proliferation ability, and the potential to repair damaged tissues, fight aging and various diseases, and have good clinical application prospects.

[0003] At present, the culture of umbilical cord mesenchymal stem cells is mainly carried out in serum-containing culture medium. Although serum-containing culture medium plays an important role in the culture of umbilical cord mesenchymal stem cells, the serum composition is complex, there is a potential risk of pathogenic biological contamination, and the differences between batches are large, which may affect the stability and reliability of the experimental results. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, one of the purposes of the present invention is to provide a 3D printed scaffold for large-scale culture of umbilical cord mesenchymal stem cells, which can create a 3D environment for cells while providing them with nutrients that promote their growth.

[0005] A second object of the present invention is to provide a method for culturing umbilical cord mesenchymal stem cells.

[0006] One of the purposes of the present invention is achieved by the following technical solution:

[0007] A 3D printed scaffold for large-scale culture of umbilical cord mesenchymal stem cells comprises a scaffold body, wherein the scaffold body is a 3D printed integrally formed structure, and cell culture wells are arranged on the scaffold body, wherein the cell culture wells are filled with a composite gel loaded with umbilical cord mesenchymal stem cells.

[0008] Furthermore, the preparation process of the composite gel is as follows: a compound containing a cyano group is reacted with a water-soluble polymer to obtain a water-soluble polymer grafted with a cyano group, and then the water-soluble polymer grafted with a cyano group, aldehyde phenylboronic acid and a polymer having multiple hydroxy groups are added into water for mixing and stirring to react, thereby obtaining a composite gel.

[0009] Furthermore, the compound containing a cyano group is one of cyanoacetic acid and ethyl cyanoacetate, the water-soluble polymer is one of polyethylene glycol, polyvinyl alcohol, and gelatin, and the reaction conditions of the compound containing a cyano group and the water-soluble polymer are heating at 85°C for 23-26h under alkaline conditions.

[0010] Furthermore, the molar concentration of the water-soluble polymer grafted with cyano groups in aqueous solution is 5%-15%; the molar concentration of the aldehyde phenylboronic acid in aqueous solution is 5%-15%; the mass concentration of the polymer with multiple hydroxyl groups in aqueous solution is 2%-10%; and the polymer with multiple hydroxyl groups is hydroxypropyl cellulose.

[0011] Furthermore, the preparation process of the stent body is as follows: using biopolymer as the basic raw material, adding a cell growth promoter, using a solvent emulsification method to prepare microspheres loaded with the cell growth promoter, and 3D printing to obtain a stent body with cell culture holes.

[0012] Furthermore, the mass ratio of the biopolymer to the cell growth promoter is 5-20:1, the biopolymer is at least one of L-polylactic acid, polycaprolactone, and polyhydroxyalkanoate, and the cell growth promoter is at least one of sodium lithium magnesium silicate, magnesium phosphate, and copper phosphate.

[0013] Furthermore, the diameter of the cell culture hole is 100-1000 μm, and the diameter of the support body is 10-100 mm.

[0014] The second object of the present invention is achieved by adopting the following technical solution:

[0015] A method for culturing umbilical cord mesenchymal stem cells, using the 3D printed scaffold for culturing.

[0016] Furthermore, the umbilical cord mesenchymal stem cell culture method comprises the following steps: mixing the umbilical cord mesenchymal stem cells and the composite gel, infusing them into the cell culture wells of the scaffold body, and then immersing the scaffold body into a culture container containing a culture medium for culture, adding a gel dissociation agent for dissociation after the culture is completed, and collecting the cells after centrifugation and washing.

[0017] Furthermore, the density of the umbilical cord mesenchymal stem cells in the composite gel is 5×10 5 -8×10 5 / mL; the gel dissociation agent is one of cysteine ​​and selenocysteine ​​or a mixture of both, the added concentration of the gel dissociation agent in the culture medium is 50-100 mg / mL, and the dissociation time is 2-20 min.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a 3D printed scaffold for large-scale culture of umbilical cord mesenchymal stem cells. The pore structure on the scaffold can optimize cell attachment, proliferation and nutrient exchange; the scaffold body and the composite gel have good umbilical cord mesenchymal stem cell compatibility and wall adhesion rate, and the composite gel has the characteristic of rapid dissociation under specific stimulation, so that cells can be collected safely and efficiently without damaging the cell state; the scaffold provides a novel and effective solution for stem cell research and clinical application, and is particularly suitable for occasions requiring large-scale cell production, and has significant social value and commercial potential.

[0020] The present invention provides a cell culture method for large-scale culture of umbilical cord mesenchymal stem cells. The culture method is simple to operate and has high universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a top view of the 3D printed scaffold for culturing umbilical cord mesenchymal stem cells according to Example 1 of the present invention, wherein 1 is a scaffold body; 11 is a cell culture hole; 12 is a composite gel;

[0022] Figure 2 Schematic diagram of the cell morphology of umbilical cord mesenchymal stem cells cultured for 3 days in Example 1, scale bar = 50 μm;

[0023] Figure 3 Schematic diagram of the cell morphology of umbilical cord mesenchymal stem cells cultured for 3 days in Comparative Example 1, scale bar = 50 μm;

[0024] Figure 4 Schematic diagram of the cell morphology of umbilical cord mesenchymal stem cells cultured for 3 days in Comparative Example 2, scale bar = 50 μm;

[0025] Figure 5 Schematic diagram of the cell morphology of umbilical cord mesenchymal stem cells cultured for 3 days in Comparative Example 3, scale bar = 50 μm. DETAILED DESCRIPTION

[0026] Below, in conjunction with the accompanying drawings and specific embodiments, the present invention is further described. It should be noted that, under the premise of no conflict, the embodiments described below or the technical features can be arbitrarily combined to form new embodiments. The specific conditions not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0027] Example 1

[0028] A 3D printed scaffold for large-scale culture of umbilical cord mesenchymal stem cells, comprising a scaffold body 1, the scaffold body 1 is a 3D printed integrated structure, a cell culture well 11 is arranged on the scaffold body 1, and a composite gel 12 loaded with umbilical cord mesenchymal stem cells is filled in the cell culture well 11, such as Figure 1 As shown;

[0029] The preparation process of the above-mentioned stent body is as follows:

[0030] (1) dissolving L-polylactic acid in chloroform to obtain a L-polylactic acid solution, and uniformly dispersing sodium lithium magnesium silicate in the L-polylactic acid solution to obtain an organic phase, wherein the mass ratio of the L-polylactic acid to the sodium lithium magnesium silicate is 5:1;

[0031] (2) Sodium dodecyl sulfate is dissolved in a saturated aqueous solution of chloroform to prepare an aqueous phase of the emulsifier, and the mass concentration of sodium dodecyl sulfate is 3%;

[0032] (3) adding the organic phase of step (1) to the aqueous phase of step (2), emulsifying into an emulsion by mechanical stirring, then removing the solvent by solvent evaporation, washing and drying to obtain L-polylactic acid microspheres loaded with sodium magnesium lithium silicate;

[0033] (4) The microspheres prepared in step (3) were used as the material and printed using an FDM 3D printer with a nozzle aperture of 200 μm, a wire spacing of 100 μm, and an interlayer angle of 45° / 90° / 135° to obtain a scaffold body with a diameter of 10 mm, a height of 10 mm, and a cell culture hole diameter of 100 μm.

[0034] The preparation process of the above composite gel is as follows:

[0035] A: Take polyvinyl alcohol (PVA2488, 4eq) and heat it to 85℃ in a reaction bottle to melt it. Then add cyanoacetic acid (CAA, 1eq) into the reaction bottle, followed by Na2CO3 (0.2eq). Continue to react at 85℃ for 23h, and purify to obtain the target product PVA-CAA.

[0036] B: The PVA-CAA, 2-formaldehyde phenylboronic acid (2-FPBA) and hydroxypropyl cellulose prepared in step A are dissolved in deionized water, the molar concentrations of PVA-CAA and 2-formaldehyde phenylboronic acid are both 10%, the mass concentration of hydroxypropyl cellulose is 2%, and then mixed to prepare a composite gel.

[0037] A method for culturing umbilical cord mesenchymal stem cells, the process is as follows:

[0038] The frozen P3 umbilical cord mesenchymal stem cells were revived and mixed with the composite gel. The density of umbilical cord mesenchymal stem cells was 5×10 5 / mL, the above composite gel was perfused into the cell culture wells of the scaffold body, and then the scaffold body was immersed in a culture container containing DMEM / F12 culture medium, and cultured in an incubator at 37°C, 5% CO2, and some culture samples were taken to detect the adhesion rate of umbilical cord mesenchymal stem cells at 24h, and the cell morphology was observed after culturing for 3d. After continuous culture for 7d, cysteine ​​was added for dissociation for 10min, and the added concentration of cysteine ​​in the culture medium was 50mg / mL. After the dissociation was completed, the umbilical cord mesenchymal stem cells were collected by centrifugation and washing.

[0039] Example 2

[0040] A 3D printed scaffold for large-scale culture of umbilical cord mesenchymal stem cells, comprising a scaffold body, the scaffold body being a 3D printed one-piece structure, a cell culture well being arranged on the scaffold body, and a composite gel loaded with umbilical cord mesenchymal stem cells being filled in the cell culture well;

[0041] The preparation process of the above-mentioned stent body is as follows:

[0042] (1) dissolving polycaprolactone in chloroform to obtain a polycaprolactone solution, and uniformly dispersing copper phosphate in the polycaprolactone solution to obtain an organic phase, wherein the mass ratio of polycaprolactone to copper phosphate is 10:1;

[0043] (2) Sodium dodecyl sulfate is dissolved in a saturated aqueous solution of chloroform to prepare an aqueous phase of the emulsifier, and the mass concentration of sodium dodecyl sulfate is 3%;

[0044] (3) adding the organic phase of step (1) to the aqueous phase of step (2), emulsifying into an emulsion by mechanical stirring, then removing the solvent by solvent evaporation, washing and drying to obtain polycaprolactone microspheres loaded with copper phosphate;

[0045] (4) The microspheres prepared in step (3) were used as the material and printed using an FDM 3D printer with a nozzle aperture of 200 μm, a wire spacing of 500 μm, and an interlayer angle of 45° / 90° / 135°. A scaffold body with a diameter of 50 mm, a height of 10 mm, and a cell culture hole diameter of 500 μm was obtained.

[0046] The preparation process of the above composite gel is as follows:

[0047] A: Take gelatin (4 eq) and heat it to 85℃ in a reaction bottle to melt it. Then add ethyl cyanoacetate (ECA, 1 eq) into the reaction bottle, followed by Na2CO3 (0.2 eq). Continue to react at 85℃ for 25 hours, and purify to obtain the target product gelatin-ECA.

[0048] B: The gelatin-ECA, 2-formaldehyde phenylboronic acid (2-FPBA) and hydroxypropyl cellulose prepared in step A are dissolved in deionized water and mixed and stirred to prepare a composite gel. The molar concentrations of gelatin-ECA and 2-formaldehyde phenylboronic acid are both 5%, and the mass concentration of hydroxypropyl cellulose is 5%.

[0049] A method for culturing umbilical cord mesenchymal stem cells, the process is as follows:

[0050] The frozen P3 umbilical cord mesenchymal stem cells were revived and mixed with the composite gel. The density of umbilical cord mesenchymal stem cells was 6×10 5 / mL, the above composite gel was perfused into the cell culture wells of the scaffold body, and then the scaffold body was immersed in a culture container containing DMEM / F12 culture medium, and cultured in an incubator at 37°C, 5% CO2, and some culture samples were taken to detect the adhesion rate of umbilical cord mesenchymal stem cells at 24h, and the cell morphology was observed after culturing for 3d. After continuous culture for 7d, selenocysteine ​​was added for dissociation for 20min, and the added concentration of selenocysteine ​​in the culture medium was 80mg / mL. After the dissociation was completed, the umbilical cord mesenchymal stem cells were collected by centrifugation and washing.

[0051] Example 3

[0052] A 3D printed scaffold for large-scale culture of umbilical cord mesenchymal stem cells, comprising a scaffold body, the scaffold body being a 3D printed one-piece structure, a cell culture well being arranged on the scaffold body, and a composite gel loaded with umbilical cord mesenchymal stem cells being filled in the cell culture well;

[0053] The preparation process of the above-mentioned stent body is as follows:

[0054] (1) dissolving polyhydroxyalkanoate in chloroform to obtain a polyhydroxyalkanoate solution, and uniformly dispersing magnesium phosphate in the polyhydroxyalkanoate solution to obtain an organic phase, wherein the mass ratio of polyhydroxyalkanoate to magnesium phosphate is 20:1;

[0055] (2) Sodium dodecyl sulfate is dissolved in a saturated aqueous solution of chloroform to prepare an aqueous phase of the emulsifier, and the mass concentration of sodium dodecyl sulfate is 3%;

[0056] (3) adding the organic phase of step (1) to the aqueous phase of step (2), emulsifying into an emulsion by mechanical stirring, then removing the solvent by solvent volatilization, washing and drying to obtain polyhydroxyalkanoate microspheres loaded with magnesium phosphate;

[0057] (4) The microspheres prepared in step (3) were used as the material and printed using an FDM 3D printer with a nozzle aperture of 200 μm, a wire spacing of 1000 μm, and an interlayer angle of 45° / 90° / 135° to obtain a scaffold body with a diameter of 100 mm, a height of 10 mm, and a cell culture hole diameter of 1000 μm.

[0058] The preparation process of the above composite gel is as follows:

[0059] A: Take polyethylene glycol (PEG3000, 4eq) and heat it to 85℃ in a reaction bottle to melt it. Then add cyanoacetic acid (CAA, 1eq) into the reaction bottle, followed by Na2CO3 (0.2eq). Continue to react at 85℃ for 26h, and purify to obtain the target product PEG-CAA.

[0060] B: The PEG-CAA, 2-formaldehyde phenylboronic acid (2-FPBA) and hydroxypropyl cellulose prepared in step A are dissolved in deionized water and mixed and stirred to prepare a composite gel. The molar concentrations of PEG-CAA and 2-formaldehyde phenylboronic acid are both 15%, and the mass concentration of hydroxypropyl cellulose is 10%.

[0061] A method for culturing umbilical cord mesenchymal stem cells, the process is as follows:

[0062] The frozen P3 umbilical cord mesenchymal stem cells were revived and mixed with the composite gel. The density of umbilical cord mesenchymal stem cells was 8×10 5 / mL, the above composite gel was perfused into the cell culture wells of the scaffold body, and then the scaffold body was immersed in a culture container containing DMEM / F12 culture medium, and cultured in an incubator at 37°C, 5% CO2, and some culture samples were taken to detect the adhesion rate of umbilical cord mesenchymal stem cells at 24h, and the cell morphology was observed after culturing for 3d. After continuous culture for 7d, cysteine ​​was added for dissociation for 2min, and the concentration of cysteine ​​added in the culture medium was 100mg / mL. After the dissociation was completed, the umbilical cord mesenchymal stem cells were collected by centrifugation and washing.

[0063] Comparative Example 1

[0064] The difference between comparative example 1 and embodiment 1 is that the bracket body is omitted, and the rest is the same as embodiment 1.

[0065] Comparative Example 2

[0066] The difference between Comparative Example 2 and Example 1 is that hydroxypropyl cellulose is not added in step B, and the rest is the same as Example 1.

[0067] Comparative Example 3

[0068] The difference between Comparative Example 3 and Example 1 is that the composite gel is omitted, the cells are resuspended in DMEM / F12 culture medium and then perfused into the cell culture wells of the scaffold body, and the rest is the same as Example 1.

[0069] Test Example 1

[0070] Some umbilical cord mesenchymal stem cells cultured for 24 hours in Examples 1-3 and Comparative Examples 1-3 were dissociated, centrifuged, washed, and then counted to obtain the number of adherent cells. Adhesion rate (%) = number of adherent cells / total number of inoculated cells × 100%. The results are shown in Table 1.

[0071] Table 1 Adhesion rate of umbilical cord mesenchymal stem cells

[0072]

[0073] It can be seen from the results in Table 1 that the 3D printed scaffold prepared in the present application can improve the adhesion rate of umbilical cord mesenchymal stem cells. The adhesion rates in Examples 1 to 3 all exceeded 90%, and the adhesion effect was better than that in Comparative Examples 1 to 3.

[0074] Test Example 2

[0075] The umbilical cord mesenchymal stem cells cultured for 3 days in Examples 1 to 3 and Comparative Examples 1 to 3 were stained with Calcein AM before gel dissociation and observed under a microscope. The results are as follows: Figures 2 to 5 As shown (scale bar = 50 μm).

[0076] Depend on Figures 2 to 5 It can be seen that compared with Comparative Examples 1 to 3, more umbilical cord mesenchymal stem cells were stained in Example 1, indicating that there were more living cells and a higher cell density in Example 1.

[0077] Test Example 3

[0078] The umbilical cord mesenchymal stem cells collected after 7 days of culture in Examples 1 to 3 and Comparative Examples 1 to 3 were counted, and the proliferation times were calculated, where the proliferation times = the total number of cells after 7 days of culture / the total number of initial cell inoculation. The results are shown in Table 2.

[0079] Table 2 Proliferation multiples of umbilical cord mesenchymal stem cells

[0080]

[0081] It can be seen from the results in Table 2 that a large number of umbilical cord mesenchymal stem cells are cultured in Examples 1 to 3, indicating that the 3D printed scaffolds of Examples 1 to 3 can better promote the proliferation of umbilical cord mesenchymal stem cells and improve cell proliferation activity.

[0082] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A 3D printed scaffold for large-scale culture of umbilical cord mesenchymal stem cells, characterized in that: It comprises a stent body, the stent body is a 3D printed integrated structure, a cell culture hole is arranged on the stent body, and the cell culture hole is filled with a composite gel loaded with umbilical cord mesenchymal stem cells; The preparation process of the composite gel is as follows: a compound containing a cyano group is reacted with a water-soluble polymer to obtain a water-soluble polymer grafted with a cyano group, and then the water-soluble polymer grafted with a cyano group, aldehyde phenylboronic acid and a polymer having multiple hydroxy groups are added into water for mixed stirring reaction to obtain a composite gel; the compound containing a cyano group is one of cyanoacetic acid and ethyl cyanoacetate, the water-soluble polymer is one of polyethylene glycol, polyvinyl alcohol and gelatin, and the polymer having multiple hydroxy groups is hydroxypropyl cellulose; The preparation process of the stent body is as follows: using biopolymer as the basic raw material, adding a cell growth promoter, using a solvent emulsification method to prepare microspheres loaded with the cell growth promoter, and 3D printing to obtain the stent body with cell culture holes; the biopolymer is at least one of L-polylactic acid, polycaprolactone, and polyhydroxyalkanoate, and the cell growth promoter is at least one of lithium magnesium sodium silicate, magnesium phosphate, and copper phosphate; The culture of the umbilical cord mesenchymal stem cells comprises the following steps: mixing the umbilical cord mesenchymal stem cells and the composite gel, and infusing them into the cell culture wells of the scaffold body, and then immersing the scaffold body into a culture container containing a culture medium for culture, adding a gel dissociation agent for dissociation after the culture is completed, and collecting the cells after centrifugation and washing; the gel dissociation agent is one or a mixture of cysteine ​​and selenocysteine.

2. The 3D printed scaffold for large-scale culture of umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The conditions for the reaction of the compound containing a cyano group and the water-soluble polymer are heating at 85° C. for 23-26 hours under alkaline conditions.

3. The 3D printed scaffold for large-scale culture of umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The molar concentration of the water-soluble polymer grafted with cyano groups in aqueous solution is 5%-15%; the molar concentration of the aldehyde phenylboronic acid in aqueous solution is 5%-15%; and the mass concentration of the polymer with multiple hydroxyl groups in aqueous solution is 2%-10%.

4. The 3D printed scaffold for large-scale culture of umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The mass ratio of the biopolymer to the cell growth promoter is 5-20:

1.

5. The 3D printed scaffold for large-scale culture of umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The diameter of the cell culture hole is 100-1000 μm, and the diameter of the support body is 10-100 mm.

6. The 3D printed scaffold for large-scale culture of umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The density of the umbilical cord mesenchymal stem cells in the composite gel is 5×10 5 -8×10 5 / mL; the added concentration of the gel dissociation agent in the culture medium is 50-100 mg / mL, and the dissociation time is 2-20 min.

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