CN117757276A - Suspension printing support material for supporting cell growth and preparation method and application thereof - Google Patents
Suspension printing support material for supporting cell growth and preparation method and application thereof Download PDFInfo
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- CN117757276A CN117757276A CN202311781459.5A CN202311781459A CN117757276A CN 117757276 A CN117757276 A CN 117757276A CN 202311781459 A CN202311781459 A CN 202311781459A CN 117757276 A CN117757276 A CN 117757276A
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Landscapes
- Materials For Medical Uses (AREA)
Abstract
The invention provides a suspension printing support material supporting cell growth, a preparation method and application thereof, wherein the suspension printing support material comprises a biological base polymer modified by a polymerizable monomer and an inorganic nano material; the bio-based polymer modified by the polymerizable monomer is matched with the inorganic nano material, so that the obtained suspension printing support material can be used for achieving excellent printability and bioactivity, preloading cells and supporting cell production, has excellent mechanical property and strong enough supporting force, is suitable for suspension printing, can be used as a suspension bath to print complex artificial vascularized organs, and provides a feasible scheme for printing hierarchical vascularized large-scale organs.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to a suspension printing support material for supporting cell growth, and a preparation method and application thereof.
Background
The 3D printing technology is used as a tissue engineering technology, and takes living cells and biological base materials as raw materials on the basis of 3D printing, so that the positioning of the biological materials, living cells, functional components and the like in each layer is accurately controlled, and the 3D bracket which can be used for transplanting is formed by printing layer by layer.
Currently, the main technologies for biomaterial accumulation and formation are inkjet printing, micro-extrusion printing, and photo-assisted printing. The heating nozzle of the temperature-controlled ink-jet printer can enable the nozzle to generate pulse air pressure to print out liquid drops, and the acoustic printer generates pulse pressing through piezoelectric type or ultrasonic type; micro-extrusion printers use pneumatic or mechanical drive systems to extrude continuous pellets or wire containing material and cells; light assisted printing utilizes light focused on an energy absorbing substrate to provide a driving force for printing biological materials; printing of vascularized structures with spatially defined features is difficult with the printing methods described above, and there are currently the following problems for constructing large scale hierarchy vascularized organs: (1) The construction material of the hierarchical blood vessel needs to be compatible with cell compatibility (especially supporting self-assembly based on endothelial cell micro-vascular network) and engineering plasticity; (2) The printing time of the large-scale organ is long, and the survival of cells cannot be supported; (3) The large-scale vascularized organ comprises a tissue structure and a vascular structure, and at least two levels of different tissue structures exist, and the current printing technology is difficult to realize multi-level construction.
Suspension 3D printing is to use suspension medium material to provide support, to extrude liquid phase material from a syringe into a support bath, and to shape a support with a complex 3D structure. The principle of operation of suspended 3D printing technology requires that the ink material must gel into filaments rapidly without spreading. The technology improves the forming stability of the 3D biological printing ink with weak mechanical strength in the printing process without additionally providing a printing support structure. The currently commonly used suspension medium materials are mainly gelatin microparticles, the size and uniformity of the microparticles directly influence the printing precision, and the preparation process of the supporting bath is complex. CN114854042a discloses a suspension medium for suspending 3D bioprinting, and a preparation method and application thereof; the suspension medium provided by the invention comprises the following components in percentage by mass: 0-10% of gellan gum, 0-10% of sulfhydrylation gellan gum and the balance of solvent; and the content of the gellan gum and the sulfhydrylation gellan gum is different and zero; the suspension medium provided by the invention has good thixotropic property and self-repairing capability, so that the spray head of the 3D biological printer can freely move in the suspension medium, and meanwhile, the integrity of a 3D biological printing structure can be maintained; the problem that natural polymer materials such as collagen, fibrin and the like can not realize self-supporting printing in the traditional printing is solved; however, the suspension medium provided by the invention has poor biocompatibility and cannot support cell production.
Therefore, in view of the above problems, there is an urgent need to develop a suspension printing support material that is excellent in mechanical properties and supports cell growth.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a suspension printing support material for supporting cell growth, and a preparation method and application thereof, wherein the suspension printing support material adopts a bio-based polymer and an inorganic nano material to match, can give consideration to printability and bioactivity, can preload cells and support cell production, has excellent mechanical properties, is suitable for suspension printing, and provides a feasible scheme for constructing large-scale-level vascularized organs.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a suspension printing support material supporting cell growth, the suspension printing support material comprising a bio-based polymer and an inorganic nanomaterial.
The suspension printing support material provided by the invention adopts a bio-based polymer and an inorganic nano material for matching; wherein the bio-based polymer has excellent biocompatibility, can be rapidly cured under photoinitiated or thermally initiated conditions, and the inorganic nanomaterial has a specific structure and charging properties; the inventor of the invention researches that the composite material formed by matching the two materials has good biocompatibility, can preload cells, has good shear thinning performance and self-supporting performance, and can be freely switched between a sol state (low viscosity) and a gel state (high viscosity) in the suspension printing process; specifically, in the suspension printing process, the viscosity of the composite material is reduced to be in a sol state under the action of shearing force, so that suspension printing is facilitated, after a printing technology is adopted, the shearing force disappears, the composite material can be quickly restored to a gel state, and subsequent solidification is facilitated to form a stable shape and structure;
in conclusion, the suspension printing support material combines the advantages of the bio-based polymer and the inorganic nano material, has excellent biocompatibility, mechanical property and printability, can perform suspension printing in a cell-loaded mold, increases the complexity of printing organs, and provides a feasible scheme for manufacturing hierarchical vascularized large-scale organs.
Preferably, the mass ratio of the bio-based polymer to the inorganic nanomaterial is 10 (4-12), such as 10:5, 10:6, 10:7, 10:8, 10:9, 10:10 or 10:11, etc.
As the preferable technical scheme of the invention, the mass ratio of the bio-based polymer to the inorganic nano material is further limited to 10 (4-12), if the relative dosage of the inorganic nano material is low, the mechanical property of the prepared suspension printing support material is poor, and further the support property is poor, and the printing performance is poor; if the relative amount of the inorganic nanomaterial is high, the prepared suspension printing support material has poor biocompatibility, and is unfavorable for cell growth.
Preferably, the bio-based polymer comprises an unmodified bio-based polymer and/or a polymer monomer modified bio-based polymer.
The term "biobased polymer" as used herein includes unmodified biobased polymers and biobased polymers modified with a polymer monomer, and the following references are intended to indicate the same meaning.
Preferably, the bio-based polymer of the unmodified bio-based polymer comprises any one or a combination of at least two of hyaluronic acid, chondroitin sulfate, heparin sulfate, dextran, alginate, cellulose, chitin, chitosan, gelatin, collagen, silk fibroin, albumin, keratin or elastin.
Preferably, the polymerizable monomer in the polymer monomer modified bio-based polymer comprises any one or a combination of at least two of methacrylate, methacrylamide, acrylate, styrene, hydroxyethyl methacrylate, lactic acid methacrylate, caprolactone methacrylate, norbornene, maleimide, vinyl sulfone, tetrazine, tyramine, catechol, azide-alkyne, imine, hydrazone or disulfide,
preferably, the polymerizable monomer modified bio-based polymer comprises any one or a combination of at least two of methacryloylated collagen, methacryloylated gelatin, methacryloylated hyaluronic acid, methacryloylated alginate, methacryloylated collagen silk protein, methacryloylated albumin, norbornene collagen, norbornene gelatin, norbornene alginate or norbornene hyaluronic acid.
Preferably, the inorganic nanomaterial includes any one or a combination of at least two of nanoclay, nano hydroxyapatite (nHAP), nano silicate (nSi), bioactive glass (BGn), or Mesoporous Silica (MSN).
Preferably, the suspension printing support material further comprises a photoinitiator.
For the suspension printing support material provided by the invention, if the adopted bio-based polymer comprises a bio-based polymer modified by a polymer monomer, and can only be formed by photo-curing later, a photoinitiator needs to be added, if the adopted bio-based polymer is an unmodified bio-based polymer, photo-curing forming or thermal curing forming can be selected later according to the requirement, and if thermal curing forming is selected, no additional photoinitiator needs to be added.
Preferably, the photoinitiator is a biocompatible photoinitiator.
Preferably, the biocompatible photoinitiator comprises a biocompatible ultraviolet photoinitiator or a biocompatible visible photoinitiator.
Preferably, the biocompatible ultraviolet initiator comprises 2-hydroxy-4- (2-hydroxyethoxy) -2-methylbenzophenone (Irgacure, I2959) and/or ethyl 2,4, 6-Trimethylbenzoyl Phenylphosphonate (TPO).
Preferably, the biocompatible visible light initiator comprises lithium phenyl-2, 4, 6-trimethylbenzoyl phosphinate (LAP).
In a second aspect, the present invention provides a method for preparing a suspension printing support material according to the first aspect, the method comprising: the suspension printing support material is obtained by conducting the bio-based polymer, the inorganic nanomaterial and optionally the photoinitiator in a solvent.
Preferably, the solvent comprises sterile water.
In a third aspect, the present invention provides an artificial vascularized organ obtained by suspension printing using the suspension printing support material and cells according to the first aspect.
The artificial vascularization organ provided by the invention is obtained by adopting suspension printing support materials and cells to match and then performing suspension printing, and further comprises a tissue structure and a vascular structure, wherein at least two stages of different tissue structures exist.
Preferably, the cells include any one or a combination of at least two of endothelial cells, fibroblasts, induced pluripotent stem cells, hepG2 cells, a549 cells, 293 cells, hela cells, MCF7 cells, CHO-K1 cells, mesenchymal stem cells, smooth muscle cells, hematopoietic stem cells, primary cells extracted from brain, heart, liver, kidney, intestinal tract, stomach, reproductive organ or muscle tissue organ.
Preferably, the bio-ink used for suspension printing includes any one or a combination of at least two of gelatin, hydroxyapatite, polylactic acid or polylactic acid-co-lactic acid.
In a fourth aspect, the present invention provides a method for preparing an artificial vascularized organ according to the third aspect, comprising the steps of:
(1) Adding cells to the suspension printing support material of the first aspect to obtain a suspension bath containing cells;
(2) And (3) performing suspension printing and solidification on the biological ink in the suspension bath containing the cells obtained in the step (1) to obtain the artificial vascularized organ.
According to the preparation method of the artificial vascularized organ, cells are firstly added into a suspension printing support material, the suspension printing support material has excellent biocompatibility, so that the activity of the cells cannot be affected, then the suspension printing support material carrying the cells is placed into a printing mold as a suspension bath, the suspension printing support material is solidified to be used as a primary printing structure, biological ink is adopted to carry out suspension printing in the suspension bath, a spiral sphere, a grid structure, a complex letter structure and other knots can be manufactured to be used as a secondary printing structure, and finally the biological ink is removed after solidification to form a vascular structure in the structure.
Preferably, the curing of step (2) is photo-curing or thermal-curing.
Preferably, the intensity of the photo-curing is 5-30 mW/cm 2 For example 6mW/cm 2 、7mW/cm 2 、8mW/cm 2 、9mW/cm 2 、10mW/cm 2 、11mW/cm 2 、12mW/cm 2 、13mW/cm 2 、14mW/cm 2 、16mW/cm 2 、18mW/cm 2 、20mW/cm 2 、22mW/cm 2 、24mW/cm 2 、26mW/cm 2 Or 28mW/cm 2 Etc.
Preferably, the time of the photo-curing is 10 to 60 seconds, for example, 20 seconds, 30 seconds, 40 seconds, 50 seconds, or the like. In a fifth aspect, the present invention provides the use of an artificial vascularised organ according to the third aspect in an organogenesis study, an organ pathology study or a drug screening study.
Compared with the prior art, the invention has the following beneficial effects:
the suspension printing support material for supporting cell growth provided by the invention comprises a bio-based polymer and an inorganic nano material; the biological base polymer and the inorganic nano material are matched, so that the obtained suspension printing support material can be compatible with excellent printability and bioactivity, can preload cells and support cell production, has excellent mechanical property, has strong enough supporting force, is suitable for suspension printing, can be used as a suspension bath to print complex artificial vascularized organs, and provides a feasible scheme for printing hierarchical vascularized large-scale organs.
Drawings
FIG. 1 is a graph of storage modulus G 'and loss modulus G' as a function of temperature for the suspension printing support material provided in example 1;
FIG. 2 is a graph of storage modulus G 'and loss modulus G' as a function of temperature for the suspension printing support material provided in comparative example 1;
FIG. 3 is a graph showing the storage modulus G' and loss modulus G″ over time for the suspension printing support material provided in example 1 under different shear stresses;
FIG. 4 is a graph showing the storage modulus G 'and loss modulus G' over time for the suspension printing support material provided in comparative example 1 under different shear stresses;
FIG. 5 is a diagram showing a structure of a grid obtained by printing using the suspension printing support material provided in example 1 as a suspension bath;
FIG. 6 is a diagram of a grid structure printed using the suspension printing support material provided in example 2 as a suspension bath;
FIG. 7 is a diagram showing a structure of a grid obtained by printing using the suspension printing support material provided in example 3 as a suspension bath;
FIG. 8 is a diagram showing the structure of a grid obtained by printing using the suspension printing support material provided in comparative example 1 as a suspension bath;
FIG. 9 is a complex letter structure diagram printed using the suspension printing support material provided in example 1 as a suspension bath;
FIG. 10 is a front view of a spiral sphere printed using the suspension printing support material provided in example 1 as a suspension bath;
FIG. 11 is a top view of a spiral sphere printed using the suspension printing support material provided in example 1 as a suspension bath;
FIG. 12 is an inverted microscopic view of HepG2 cells cultured on days 0, 1, 3, and 5 in the suspension printing support material provided in example 1;
FIG. 13 is an inverted microscopic view of HepG2 cells cultured on days 0, 1, 3, and 5 in the suspension printing support material provided in comparative example 1;
FIG. 14 is a graph showing the trend of cell activity of HepG2 cells after culturing days 1, 3 and 5 in the suspension printing support material provided in examples 1 to 3 and comparative example 1;
FIG. 15 is a budding plot of endothelial cells on the day of culture, after 1 and after 2 in the suspension printing support material provided in example 1;
FIG. 16 is a budding plot of endothelial cells after day 1 and 2 of culture in the suspension printing support material provided in comparative example 1.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The experimental methods in the following embodiments are conventional methods unless otherwise specified.
Materials, reagents and the like used in the following embodiments are commercially available unless otherwise specified.
The partial raw material information used in the following embodiments is as follows:
methacryloylated collagen (ColMA): purchased from sumac Yongqin intelligent devices limited;
phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate (LAP): purchased from sumac Yongqin intelligent devices limited;
nanoclay (XLS): purchased from BYK corporation;
phosphate Buffered Saline (PBS): purchased from Gibco;
HepG2 cells: purchased from Servicebio corporation;
HUVEC cells: purchased from middling Qiao Xinzhou;
endothelial cell EGM-2 Medium set: purchased from Lonza;
gelatin: purchased from Sigma;
0.05% trypsin-EDTA and phenol red: purchased from Gibco;
composition of HepG2 cell culture medium: comprises MEM (purchased from Servicebio) and fetal bovine serum (purchased from Sigma) in a volume ratio of 9:1, and penicillin-streptomycin (purchased from Bio-Channel 7) in a volume percentage of 1%;
cell activity detection reagent: purchased from norvain.
Example 1
A suspension printing support material for supporting cell growth, the preparation method comprises the following steps:
(1) Dissolving methacryloylated collagen (ColMA) and 10 XPBS buffer solution (pH 7.2) in sterile water to obtain ColMA mother liquor with mass percent of 10%;
dissolving nanoclay (XLS) and 10 XSBS buffer (pH value is 7.2) in sterile water to obtain XLS mother liquor with the mass percentage content of 8%;
(2) Mixing the ColMA mother liquor obtained in the step (1) and the XLS mother liquor according to the volume ratio of 1:1, and finally adding the lithium 2,4, 6-trimethylbenzoyl phosphite (LAP) (so that the concentration of the LAP is 0.125g/100 mL), and fully blowing and uniformly mixing to obtain the suspension printing support material.
Examples 2 to 3
A suspension printing support material supporting cell growth, which is different from example 1 in that the mass percentage of XLS in the XLS mother liquor in step (1) is 4% (example 2) and 12% (example 3), respectively, and other substances, conditions and steps are the same as those in example 1.
Comparative example 1
A suspension printing support material, the method of making comprising: colMA and 10 XPBS buffer (pH 7.2) were dissolved in sterile water to give a mass percent of 5% ColMA as a direct support material for suspension printing.
Performance test:
(1) Rheological properties: the rheological properties of the suspension printing support material were measured at room temperature using an MCR 302 advanced rotary rheometer (Anton Paar, graz, austria);
(1) and (3) temperature scanning: selecting a parallel plate rotor, wherein the gap between the rotor and a bottom plate of the rheometer is required to be controlled to ensure that the test is performed under good contact, when the temperature scanning test is performed, a small amount of material to be tested is added in the center of a lower plate of the rheometer, then 405nm blue light irradiation is adjusted, the illumination intensity is 20mW, the illumination time is 20s, after photo-curing, the rotor is immediately controlled to descend to contact the surface of a sample to generate a bonding effect, the gap between the sample and the lower plate is about 3mm, the gap value can be slightly adjusted according to the actual thickness of the sample in the actual process, the good contact between the sample and a sensor on the rotor is ensured, the temperature rising rate is adjusted to be 4 ℃/min in an oscillation shear scanning mode with the fixed frequency of 1rad/s and the strain of 1%, the temperature is set to be 0-40 ℃ interval range to be increased, and the change of the storage modulus G 'and the loss modulus G' along with the temperature is recorded;
(2) shear repair scan: performing a shear recovery test by using a similar change shear strain of 10-1000%, performing detailed operation at the same temperature, and recording the change of the storage modulus G 'and the loss modulus G' with time under different shear stresses;
the suspension printing support materials provided in example 1 and comparative example 1 were tested according to the above test methods, respectively, to obtain the graph of change of the storage modulus G 'and the loss modulus G "of the suspension printing support material provided in example 1 with respect to temperature as shown in fig. 1, to obtain the graph of change of the storage modulus G' and the loss modulus G" of the suspension printing support material provided in comparative example 1 with respect to temperature as shown in fig. 2, to obtain the graph of change of the storage modulus G 'and the loss modulus G "of the suspension printing support material provided in example 1 with respect to time as shown in fig. 3, and to obtain the graph of change of the storage modulus G' and the loss modulus G" of the suspension printing support material provided in comparative example 1 with respect to time as shown in fig. 4;
as can be seen from fig. 1 and 2, the suspension printing support material provided in example 1 exhibited a gel transition point at about 30.06 ℃, whereas the suspension printing support material provided in comparative example 1 exhibited no gel transition point throughout.
As can be seen from fig. 3 and 4, the periodic variation of the storage modulus G 'and the loss modulus G "of the suspension printing support material provided in example 1 indicates that the suspension printing support material provided in example 1 has the shear repairing property, while the non-periodic variation of the storage modulus G' and the loss modulus G" of the suspension printing support material provided in comparative example 1 indicates the non-shear repairing property;
in conclusion, the suspension printing support material provided by the invention has good shear thinning performance and self-supporting performance, can be freely switched between a sol state (low viscosity) and a gel state (high viscosity), and is suitable for being used as a suspension bath for suspension printing.
(2) Suspension printing Performance
(1) Adding a suspension printing support material into a mould of a suspension printer, and placing the suspension printing support material on a printing platform of the suspension printer; preparing 20% gelatin (PBS buffer solution as solvent), adding color dye (conventionally sold in market), heating in a 65 ℃ oven to melt, mixing, and adding appropriate amount of gelatin solution into a printing cylinder to serve as biological ink;
(2) generating a G code according to the STL file of the three-dimensional model by using standard software, and transmitting a command to each layer of printing motion;
(3) using a 3D biological printer to take a suspension printing supporting material as a suspension bath, setting the temperature of a charging barrel and the temperature of a platform in advance, waiting until the suspension bath and the biological ink are balanced to the set temperature, extruding the biological ink into the suspension printing supporting material through a 23G needle head with the inner diameter of 25mm and the tube length of 25mm for printing, and manufacturing a spiral sphere, a grid-shaped structure and a complex letter structure;
wherein, the printer is set up as follows when printing the grid-like structure: the temperature of the platform is set to 10 ℃, the temperature of the charging barrel is set to 28 ℃, the temperature of the needle point is set to 28 ℃, the extrusion speed is 20mm/s, the air pressure is 0.14MPa, the layer height is 0.75mm, and the filling interval is 1mm;
the printer settings were as follows when printing the spiral sphere structure: the temperature of the platform is set to 15 ℃, the temperature of the charging barrel is set to 28 ℃, the temperature of the needle point is set to 28 ℃, the extrusion speed is 4mm/s, the air pressure is 0.14MPa, the layer height is 1mm, and the filling interval is 1mm;
the printer settings when printing the letter structure were as follows: the temperature of the platform is set to 15 ℃, the temperature of the charging barrel is set to 27 ℃, the temperature of the needle point is set to 27 ℃, the extrusion speed is 4mm/s, and the air pressure is 0.18MPa;
according to the printing method, suspension printing is carried out by using the suspension printing support materials provided in the embodiments 1-3 and the comparative example 1 as suspension baths, wherein grid structure diagrams obtained by using the suspension printing support materials provided in the embodiments 1-3 as suspension baths are shown in fig. 5-7, and grid structure diagrams obtained by using the suspension printing support materials provided in the comparative example 1 as suspension baths are shown in fig. 8; the complex letter structure obtained by using the suspension printing support material provided in example 1 as a suspension bath is shown in fig. 9; a front view and a top view of a spiral sphere printed using the suspension printing support material provided in example 1 as a suspension bath are shown in fig. 10 and 11, respectively;
as can be seen from fig. 5 to 11, the suspension printing support materials provided in examples 1 to 3 have good printing properties, wherein example 1 has the best printing properties, and can support accurate grid, curve printing, and letter printing, whereas the suspension printing support material provided in comparative example 1 cannot support accurate grid structure printing, indicating that it has no printing properties.
(3) Relative quantitative test experiment of hepG2 cell Activity
(1) Culturing HepG2 cells: culturing HepG2 cells by using a T75-specification culture bottle, adding 8mL MEM culture medium into each T75, replacing the culture medium every other day, observing that cell fusion reaches 90% by using an inverted microscope to obtain passages, removing the original culture medium, washing the cells once by using 2mL PBS buffer solution, adding 2mL pancreatin into each bottle of cells, placing the cells in a 37 ℃ culture box for digestion for 3min, taking out the cells, adding 2mL MEM culture medium to stop digestion after observing that cells at the bottom of the bottle fall off by naked eyes, collecting the cells into a 15mL centrifuge tube (400 g,5 min), taking out supernatant, re-suspending the cells by using a proper amount of culture medium, taking 10 mu L of cell suspension for loading by an automatic cell counter to obtain cell density, and inoculating the cells with the density of 1-5 multiplied by 10 6 Calculating the diluted cell ratio, separating out a cell suspension with corresponding volume according to the required cell amount, and centrifuging again;
(2) uniformly mixing a suspension printing support material with HepG2 cells calculated according to the cell density, inoculating the mixture to a 96-well cell culture plate, adding 5 mu L of mixed cell suspension into each well, irradiating with blue light (405 nm), wherein the irradiation energy is 20mW, the irradiation time is 20s, adding 200 mu L of cell culture medium into each well after photocuring, culturing in a 5% carbon dioxide incubator at 37 ℃, replacing the cell culture medium every other day, observing the proliferation condition of the cells under an inverted microscope on the 0 th, 1 th, 3 th and 5 th days of culturing, photographing and recording, and then using the cells for detecting cell activity experiments;
(3) sample preparation of standard curve: cell densities in hepG2 cell suspensions cultured on the day of digestion were counted using a cytometer, and the cell suspensions were diluted in an equal ratio using medium: 10. 10 (10) 2 、10 3 、10 4 、10 5 Usually 5-7 concentration gradients are required, 3 multiplex wells per group, each group of cell dilutions is added to 96 well plates, 100 μl of medium is added per well and left at room temperature;
(4) preparing a sample to be tested: the cell culture plate to be tested can be taken out in advance from the incubator and placed at room temperature for 30min to balance the temperature of the culture plate to room temperature, the CellCounting-Lite3D which is equal in volume with the cell culture to be tested and is balanced to room temperature is added, for example, when a 96-hole culture plate is used, 100 mu L of CellCounting-Lite3D is sucked and added into 100 mu L of the cell culture to be tested, and the cell mass is fully cracked by intense shaking for 5min, and the cell cracking step in the experimental process can be always placed in a horizontal shaking table for shaking incubation for 2h in a dark place to stabilize a luminous signal, so that detection can be performed.
(5) And (3) preparing a standard curve, preparing a standard curve which takes the cell number as an X-axis coordinate and the luminescence value as a Y-axis coordinate according to the known cell quantity and the luminescence detection value of the corresponding group, calculating the number of each group of living cells of the experimental data of the current day according to the standard curve of each day, and carrying out statistical analysis on the number data of the living cells of three days.
The suspension printing support materials provided in examples 1 to 3 and comparative example 1 were tested according to the above-described test methods, the inverted microscopic images of the HepG2 cells cultured in the suspension printing support material provided in example 1 at days 0, 1, 3 and 5 were shown in fig. 12, the inverted microscopic images of the HepG2 cells cultured in the suspension printing support material provided in comparative example 1 at days 0, 1, 3 and 5 were shown in fig. 13, and the cell activity trend charts of the HepG2 cells cultured in the suspension printing support materials provided in examples 1 to 3 and comparative example 1 after culturing for days 1, 3 and 5 were shown in fig. 14 after calculation;
as can be seen from fig. 12 to 14, the suspension printing support materials provided in examples 1 to 3 have good biological activity, support continuous growth of cells, and are not much different from the cell activity in the suspension printing support material provided in comparative example 1, which indicates that the addition of nanoclay does not affect the cell activity.
(4) Endothelial cell mass budding experiment
(1) Vascular Endothelial Cells (HUVEC) were cultured: culturing HUVEC cells by using culture bottles of T75 specifications, adding 8mL of EGM-2 culture medium into each T75, replacing the culture medium every day, observing that cell fusion reaches 90% by using an inverted microscope to obtain passages, removing the original culture medium, washing the cells once by using 2mL of PBS buffer solution, adding 2mL of pancreatin into each bottle of cells, placing the cells in a 37 ℃ incubator to digest for 3min, taking out the cells, adding 2mL of EGM-2 culture medium to terminate digestion after the cells at the bottom of the bottle are observed to fall off visually, collecting the cells into a 15mL centrifuge tube (400 g,5 min), taking out supernatant, re-suspending the cells by using a proper amount of culture medium, and taking 10 mu L of cell suspension for loading by an automatic cell counter to obtain the cell density; according to the cell density, calculating the dilution ratio, inoculating the cell suspension into a pretreated 96-hole transparent round bottom ultra-low adsorption micro-pore plate along the pore wall, supplementing about 200 cells in each hole with a cell culture solution until the total volume reaches about 100 mu L in each hole, and if the culture plate cannot be vigorously shaken or shake-cultured, the ultra-low adsorption water mucilage glue can be damaged, so that the cells cannot be agglomerated; placing the culture plate with the cells in a 5% carbon dioxide incubator at 37 ℃ for culturing, and starting agglomerating after 12 hours in general;
(2) agglomeration experiment: preparing a suspension printing preparation material as a precursor solution, mixing the endothelial cell pellets agglomerated within 12-24 h of culture into the precursor solution, then adding 500 mu L of the precursor solution containing the cell pellets into a cell culture dish, curing by blue light irradiation for 20s, adding 1mL of culture medium into each dish, then placing the dishes into a 37 ℃ and 5% carbon dioxide incubator for culture, changing the culture medium every day, placing the dishes under an inverted microscope for 1 and 2 days of culture to observe the sprouting condition of the endothelial cell pellets, and taking a photo and recording.
The suspension printing support materials provided in example 1 and comparative example 1 were tested according to the above-described test method, and the budding charts of endothelial cells on the day of culture, after culture 1 and after culture 2 in the suspension printing support material provided in example 1 are shown in fig. 15, and budding charts of endothelial cells on the day of culture, after culture 1 and after culture 2 in the suspension printing support material provided in comparative example 1 are shown in fig. 16;
as can be seen from fig. 15 and 16, the suspension printing support material provided in example 1 has good vascularization activity, supporting spontaneous sprouting of endothelial cells in example 1.
The applicant states that the present invention, by way of the above examples, illustrates a suspension printing support material supporting cell growth and methods of making and using the same, but the present invention is not limited to, i.e., does not necessarily rely on, the above process steps to practice the present invention. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.
Claims (10)
1. A suspension printing support material supporting cell growth, wherein the suspension printing support material comprises a bio-based polymer and an inorganic nanomaterial.
2. The suspension printing support material according to claim 1, wherein the mass ratio of the bio-based polymer to the inorganic nanomaterial is 10 (4-12);
preferably, the bio-based polymer comprises an unmodified bio-based polymer and/or a polymer monomer modified bio-based polymer;
preferably, the unmodified bio-based polymer comprises any one or a combination of at least two of hyaluronic acid, chondroitin sulfate, heparin sulfate, dextran, alginate, cellulose, chitin, chitosan, gelatin, collagen, silk fibroin, albumin, keratin, or elastin; preferably, the polymerizable monomers in the polymer monomer modified biobased polymer include any one or a combination of at least two of methacrylate, methacrylamide, acrylate, styrene, hydroxyethyl methacrylate, lactic acid methacrylate, caprolactone methacrylate, norbornene, maleimide, vinyl sulfone, tetrazine, tyramine, catechol, azide-alkyne, imine, hydrazone, or disulfide;
preferably, the polymerizable monomer modified bio-based polymer comprises any one or a combination of at least two of methacryloylated collagen, methacryloylated gelatin, methacryloylated hyaluronic acid, methacryloylated alginate, methacryloylated collagen silk protein, methacryloylated albumin, norbornene collagen, norbornene gelatin, norbornene alginate or norbornene hyaluronic acid.
3. The suspension printing support material of claim 1 or 2, wherein the inorganic nanomaterial comprises any one or a combination of at least two of nanoclay, nano-hydroxyapatite, nano-silicate, bioactive glass, or mesoporous silica.
4. A suspended print support material according to any one of claims 1 to 3, further comprising a photoinitiator;
preferably, the photoinitiator is a biocompatible photoinitiator;
preferably, the biocompatible photoinitiator comprises a biocompatible ultraviolet photoinitiator or a biocompatible visible photoinitiator;
preferably, the biocompatible ultraviolet initiator comprises 2-hydroxy-4- (2-hydroxyethoxy) -2-methylbenzophenone and/or ethyl 2,4, 6-trimethylbenzoyl phenylphosphonate;
preferably, the biocompatible visible light initiator comprises lithium phenyl-2, 4, 6-trimethylbenzoyl phosphinate.
5. A method of producing the suspension printing support material according to any one of claims 1 to 4, comprising: mixing the bio-based polymer, the inorganic nano-material and optionally the photoinitiator in a solvent to obtain the suspension printing support material.
6. An artificial vascularized organ obtained by suspension printing using the suspension printing support material and cells according to any one of claims 1 to 4.
7. The artificial vascularized organ of claim 6, wherein the cells comprise any one or a combination of at least two of endothelial cells, fibroblasts, induced pluripotent stem cells, hepG2 cells, a549 cells, 293 cells, hela cells, MCF7 cells, CHO-K1 cells, mesenchymal stem cells, smooth muscle cells, hematopoietic stem cells, primary cells extracted from brain, heart, liver, kidney, intestinal tract, stomach, reproductive organ, or muscle tissue organ;
preferably, the bio-ink used for suspension printing includes any one or a combination of at least two of gelatin, hydroxyapatite, polylactic acid or polylactic acid-co-lactic acid.
8. A method of preparing an artificial vascularized organ according to claim 6 or 7, comprising the steps of:
(1) Adding cells to the suspension printing support material of any one of claims 1 to 4 to obtain a suspension bath containing cells;
(2) And (3) performing suspension printing in the suspension bath containing cells obtained in the step (1) by using biological ink, and curing to obtain the artificial vascularized organ.
9. The method of claim 8, wherein the curing of step (2) is photo-curing or thermal-curing;
preferably, the intensity of the photo-curing is 5-30 mW/cm 2 ；
Preferably, the time of the photo-curing is 10 to 60 seconds.
10. Use of the artificial vascularized organ according to claim 6 or 7 in an organogenesis study, an organ pathology study or a drug screening study.
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