CN117535223A - Brain glioma cell derivative applied to in vitro three-dimensional vascularization as well as preparation method and application thereof - Google Patents
Brain glioma cell derivative applied to in vitro three-dimensional vascularization as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a brain glioma cell derivative applied to in-vitro three-dimensional vascularization, and a preparation method and application thereof, wherein the brain glioma cell derivative comprises the following components: manufacturing a channel-patterned PDMS chip a; manufacturing a PDMS film b, and thermally bonding the PDMS film b with the surface of the chip a where the channel pattern is located; plasma treating the film b, and bonding one side and the surface of the film b with the substrate c after plasma treatment to form a chip d; filling hydrogel into the channel d of the chip, and removing the fibrin solution after dressing; filling cells derived from cerebral microvessels and growth factors into a channel of the chip d, placing the cells in an incubator, and peeling the chip a after the cerebral microvessels are attached to the bottom surface of the channel; the culture solution is not subjected to cell pattern, and a patterned vascular network is formed after culture; and pouring hydrogel containing glioma cells, proteins, growth factors and medicines on the vascular network. The vascular network can realize the nutrition supply of glioma cell derivatives and carry away cell excretions.
Description
Technical Field
The invention belongs to the technical field of tissue engineering, and particularly relates to an in-vitro three-dimensional vascularized brain glioma cell derivative and a preparation method thereof.
Background
Cell-cell, cell-microenvironment interactions play a critical role in inducing glioma cell functional expression. Natural tissues and organs have a high degree of organization and possess a complex vascular system of multiple dimensions. The multiscale vascular system can transport nutrients and oxygen to surrounding cells, carrying away cellular excretions. Engineering materials with good biocompatibility are required for the construction of in vitro physiological, pathological and pharmacological models, and the engineering materials simulate natural extracellular matrixes and microvascular systems. Soft lithography, casting, bio-spinning, bio-3D printing, microfluidic patterning, etc. have been applied to develop 2D or 3D cell-containing structures, and these microfabrication techniques can produce various multi-gradient, multi-material, multi-style biological models. However, multi-scale complex vascularized network construction within larger volumes of tissue remains challenging, adequate nutrient supply cannot be achieved within biological models, and guiding long-term cell growth within environmentally restricted spaces remains a challenge.
The current 3D bioprinting technology adopts a mode of stacking cells layer by layer in the aspect of printing blood vessels, and then constructing blood vessels in artificial tissues through subsequent biological culture. The vascular cells manufactured by the method have low density and disordered distribution, are not easy to control accurately, and meanwhile, the vascular printing forming method has the problems of low forming speed, low efficiency, poor finished product structure, incapability of forming a multi-scale complex vascular system and the like.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a brain glioma cell derivative applied to in-vitro three-dimensional vascularization, a preparation method and application thereof, a planar multi-scale vascular network is manufactured by adopting a microfluidic pattern method and a surface microstructure technology, an in-vitro physiological, pathological and pharmacological model is provided with a brain glioma microenvironment similar to vascularization in a living body, and a personalized tissue engineering vascular network brain glioma biological model is obtained, so that the problem of full vascularization in a large tissue is well solved.
The invention is realized by the following technical scheme: a method for preparing a brain glioma cell derivative applied to in vitro three-dimensional vascularization, comprising the following steps:
(1) Manufacturing a channel-patterned PDMS chip a;
(2) Manufacturing a PDMS film b, and thermally bonding the PDMS film b with the surface of the PDMS chip a where the channel pattern is located;
(3) Carrying out plasma treatment on one side of the chip with the film b, and bonding one side of the film b with a substrate c of which the surface is subjected to the plasma treatment to form a chip d;
(4) Filling hydrogel which is conducive to vascularization of endothelial cells into the channel of the chip d, placing the hydrogel into an incubator for dressing, and removing the hydrogel;
(5) Filling cells derived from cerebral microvessels and related growth factors into a channel of the chip d, placing the cells in an incubator, and peeling off the PDMS chip a after the cerebral microvessel cells are attached to the bottom surface of the channel;
(6) The culture solution is soaked in the cell pattern on the chip, and then placed in an incubator for culturing for a period of time, and the culture solution is removed to form a patterned multi-scale hierarchical vascular network;
(7) And pouring a hydrogel containing the brain glioma cells, the protein, the growth factors and the medicines on the vascular network to form a brain glioma cell derivative, wherein the vascular network can provide nutrition and oxygen for physiological, pathological and pharmacological models of the hydrogel containing the brain glioma cells and take away cell excreta.
The invention combines the microfluid pattern technology and the surface microstructure manufacturing technology to manufacture the hierarchical multi-scale protein, hydrogel and cell patterns, and can form micro-scale morphological features on a substrate and limit cells and guide the directional growth of the cells.
In one embodiment of the present invention, the step (1) further includes a step of performing hydrophilic treatment on the channel of the PDMS chip a.
Preferably, the hydrophilic treatment includes, but is not limited to, ultraviolet ozone treatment, polylysine packageQuilt and N 2 、NH 3 、O 2 One or more combination treatments of the gas plasma treatments are also included as are other treatments that achieve the same hydrophilic effect.
In one embodiment of the present invention, the method further comprises salinization treatment of the surface of the pattern of the PDMS chip a to reduce the adhesion between the PDMS chip a and the PDMS film b.
Preferably, the surface of the chip a pattern is subjected to salinization by adopting a chemical vapor deposition method.
In chemical vapor deposition, the precursor gas is the starting material in the CVD process, and its choice depends on the desired deposition material, typically an organic or inorganic compound, preferably fluorosilane or triphenylfluorosilicone.
In one embodiment of the present invention, the method for manufacturing the PDMS film b in the step (2) is spin coating, the rotation speed of the spin coater is controlled to be 1000 rpm-3000 rpm for 5-30 min, and then the PDMS film b is thermally bonded to the surface of the PDMS chip a where the channel pattern is located.
Preferably, the thickness of the PDMS film b is 2 to 10 μm, preferably 4 to 6 μm, for example 5 μm.
Preferably, the gas used in the plasma treatment is N 2 、NH 3 、O 2 。
Preferably, the substrate c is a glass sheet.
In one embodiment of the invention, the hydrogel that facilitates vascularization of endothelial cells comprises a mixed solution of one or more of fibronectin, fibrinogen, martigel.
For example, the hydrogel is a fibronectin solution with a concentration of 14-16 μg/mL. The fibronectin solution is infused to create a good brain microvascular cell growth microenvironment, and any substance that is equally potent as fibronectin is optional.
Preferably, in the step (4) and the step (5), the time of the incubation in the incubator is 1-2 hours.
In one embodiment of the present invention, the cells of the brain microvessels may be primary cells extracted from human brain or cells of brain microvessels differentiated from ipscs, including necessary cells such as brain microvessel endothelial cells, brain microvessel pericytes, etc.
In one embodiment of the invention, the growth factor comprises one or more of vEGF, EGF, FGF, hEGF and hFGF in admixture; for example, basic fibroblast growth factor and/or vascular endothelial growth factor.
In one embodiment of the invention, the density of glioma cells in the hydrogel body comprising glioma cells, protein, growth factor and drug is 10 6 ～10 8 The elasticity model of the hydrogel is 100 kPa-1000 kPa.
In one embodiment of the invention, the protein is one or more of collagen, hyaluronic acid, extracellular matrix extracted from brain tissue, neurotransmitter receptor protein (Neurotransmitter Receptor Proteins), neuronal structural protein (Neuronal Structural Proteins), synaptoprotein (Synaptic Proteins), nerve growth factor (Neurotrophic Factors), ion channel protein (Ion Channel Proteins), neuron-specific enolone reductase (AKR 1C 1), synaptosin (Synapsin), neuroinflammation and immune-Related protein (Neuroinflammatory and Immune-Related Proteins), beta amyloid protein (beta-amyoid), alpha-synuclein (alpha-synuclein), ganglion protein (neurogenin), neuronal regulatory protein (Neuronal Regulatory Proteins), neuronal conduction protein (Neuronal Transport Proteins), dynein (dynin) and actin, neuronal endocrine protein (Neuronal Endocrine Proteins), brain-derived lipoprotein (apolipoprotein), neuronal deoxyribonucleotide protein (neuronic DNA-binding protein), zinc finger protein (Zinc Finger Proteins), neurotransmitter synthase (Neurotransmitter Synthetic Enzymes), protein kinase (C (Protein Kinase C)) and kinase enzyme (A (Protein Kinase A)).
In one embodiment of the invention, the agent is one or more of an EGFR (epidermal growth factor receptor) inhibitor, PI3K/AKT/mTOR signaling pathway inhibitor, angiogenesis inhibitor, chemotherapeutic agent, immunotherapeutic (including but not limited to immune checkpoint inhibitor, CAR-T cell therapy, immune vaccine, immune cell therapy, viral immunotherapy, and immunostimulant) agent.
The invention also provides a brain glioma cell derivative applied to in-vitro three-dimensional vascularization, which is obtained by adopting the preparation method of the brain glioma cell derivative.
In one embodiment of the invention, the brain glioma cell derivative comprises a substrate, a patterned vascular network on the substrate, and a brain glioma covering the patterned vascular network.
The invention also provides application of the brain glioma cell derivative applied to in-vitro three-dimensional vascularization in medical research, in particular application in disease mechanism research, drug screening, treatment method development, radiotherapy research, vaccine research, gene editing and gene knockout research and tumor microenvironment research.
The invention has the beneficial effects that:
1) The invention provides a brain glioma cell derivative applied to in-vitro three-dimensional vascularization and a manufacturing method thereof, wherein a planar multi-scale vascular network (for example, a microfluidic pattern method and a surface microstructure technology are adopted specifically) is manufactured, a layer of three-dimensional glioma cell derivative (glioma cells + hydrogel + growth factors + proteins + drugs) is poured on the constructed vascular network pattern, and the patterned multi-scale hierarchical blood vessel can provide nutrition and oxygen for physiological, pathological and pharmacological models of the glioma cell three-dimensional derivative, so that nutrition supply of the glioma cell derivative is realized, and cell excreta can be taken away. The method has good compatibility, simple manufacture, low cost and strong universality.
2) Based on the chip channel orientation and constraint principle, the method simulates the microenvironment generated by in vivo vascularization to obtain glioma cell derivatives containing personalized tissue engineering vascular networks. The constructed multi-scale vascularization network can better solve the problem of full vascularization in a large tissue, provides a biological in-vivo vascularization-approximated glioma microenvironment for in-vitro physiological, pathological and pharmacological models, obtains a personalized tissue engineering vascular network glioma biological model, and can be applied to disease mechanism research, drug screening, treatment method development, radiation therapy research, vaccine research, gene editing and gene knockout research and tumor microenvironment research.
Drawings
FIG. 1 is a flow chart of the production of PDMS chip a according to embodiment 1 of the present invention;
FIG. 2 is a schematic diagram of the process of preparing a salinized layer by Chemical Vapor Deposition (CVD) in example 1 of the invention;
FIG. 3 is a schematic diagram of the structure of a PDMS film b fabricated by spin coating in example 1 of the present invention;
FIG. 4 is a schematic diagram showing the bonding effect of the PDMS chip a and the PDMS film b according to the embodiment 1 of the present invention;
FIG. 5 is a schematic diagram of the structure of a PDMS film b treated with a particle body such as oxygen in example 1 of the present invention;
FIG. 6 is a schematic diagram of the structure of the chip d according to the embodiment 1 of the present invention after filling the cells of the cerebral microvasculature;
FIG. 7 is a schematic diagram of the structure of the PDMS chip a separated from the chip d according to embodiment 1 of the present invention;
FIG. 8 is a graph of a multi-scale cerebrovascular network model formed on a glass substrate in example 1 of the present invention;
FIG. 9 is a schematic view showing the effect of pouring a layer of three-dimensional glioma cell derivatives onto a pattern of a cerebrovascular network in example 1 of the present invention.
FIG. 10 is a schematic structural diagram of a mask plate used in embodiment 1 of the present invention;
fig. 11 is a schematic diagram of a hierarchical multi-scale cerebrovascular network implemented by the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Example 1
(1) Channel-patterned PDMS chip a manufactured by soft lithography
Preparing a soft lithography processing template by using a wafer:
taking a wafer, cleaning the surface, then arranging a layer of photoresist SU-8 on the wafer, arranging a mask plate (white is a light-transmitting part and black is an opaque part as shown in fig. 10) on the photoresist SU-8, and drying at 95 ℃ for 9min. The nitrogen was turned on, the exposure time was set to 18s, photolithography was performed using ultraviolet irradiation, and then baked at 95℃for 6min. Developing with developer, washing with ethanol, drying with nitrogen, and hardening at 150deg.C for 30min. The photoresist SU-8 adopted in the steps is negative photoresist, is cured after being irradiated by ultraviolet light, and is baked after being cleaned by SU-8 developing solution.
Photoresist SU8-2100 spin coating (above) was used again and then baked at 95℃for 1h. After the upper layer and the lower layer are aligned, setting exposure time to 20s, baking for 10min at 95 ℃, developing again by using a developing solution, and then hardening for 30min at 150-180 ℃ to obtain the soft lithography processing template.
Preparing a PDMS chip a and performing oxygen plasma treatment:
1. and placing the prepared soft photoetching processing template into a closed culture dish with the diameter of 100mm, and then adding trimethylchlorosilane into a container, wherein the surface of the template is subjected to hydrophobic treatment after the trimethylchlorosilane volatilizes.
2. The A adhesive and the B adhesive (brand: dow Corning, model: SYLGARD 184) of Polydimethylsiloxane (PDMS) are mixed according to the mass ratio of 10:1-5:1, and the PDMS adhesive with the total weight of 15-20 g is obtained.
3. And pouring PDMS glue on the soft photoetching processing template subjected to the hydrophobic treatment, and placing the soft photoetching processing template into a vacuum drying box to remove bubbles.
4. Placing into an oven, and baking at 80 ℃ for 0.5-1h.
5. And (5) after cooling to normal temperature, removing the patterned PDMS from the template to obtain the PDMS chip a.
(2) The surface of the PDMS chip a is hydrophobic, one side of the channel of the chip a is treated by oxygen plasma for 1min with the power of 100-500W, and the oxygen plasma treatment can increase the adhesion capacity of the surface of the patterned PDMS structure, so that the patterned PDMS structure has hydrophilic characteristics.
(3) As shown in fig. 2, the surface of the chip channel side was salted by chemical vapor deposition, so that the PDMS chip a was easily peeled off in step (8).
(4) As shown in fig. 3, a PDMS film b was prepared by spin coating, the spin speed of the spin coater was controlled to 1000rpm to 3000rpm, and the spin coater was rotated for 5 to 30 minutes so that the thickness of the PDMS film b was about 5 μm, and then the PDMS film b was thermally bonded to the surface of the PDMS chip a where the channel pattern was located, to obtain the structure shown in fig. 4.
(5) The other surface of the PDMS film b was subjected to plasma treatment (power 100 to 500W, treatment time 1 min) by oxygen plasma.
(6) The chip film b and the glass sheet c whose surface has been subjected to plasma treatment are bonded to form a chip d as shown in fig. 6. A solution of fibronectin (15. Mu.g/mL) was poured into the channel d of the chip, incubated in an incubator for 2 hours, and then the fibrin solution was removed with sterilized gauze, dust-free absorbent paper, pipette, etc.
(7) And continuously filling human cerebral microvascular cells, human cerebral perivascular cells and cell growth factors (vEGF, FGF and EGF) into the channel of the chip d, culturing in an incubator, and peeling off the PDMS chip a after the cerebral microvascular cells are attached to the bottom surface of the channel for about 2 hours, as shown in figure 7.
(8) The culture solution was used to soak the cell pattern on the chip (after peeling off the PDMS chip a) and then placed in an incubator for culture, forming a patterned tissue engineering vascular network, as shown in fig. 8 and 11.
(9) After the vascular network was further cultured for 3 days, the culture solution was removed, and a cell containing brain glioma was cast on the vascular network (10 6 ～10 8 cells/mL), proteins, growth factors (e.g., vEGF (1 ng/mL-50 ng/mL), EGF (1 ng/mL-100 ng/mL), FGF (1 ng/mL-100 ng/mL)), drug hydrogel bodies (elastic modulus 100 kPa-1000 kPa), and a patterned hierarchical multi-scale cerebrovascular network was obtained, as shown in FIG. 9.
FIG. 11 is a hierarchical multiscale cerebrovascular network with channel widths of 300 μm,200 μm,150 μm and 75 μm for PDMS chip a in order.
According to the invention, a layer of three-dimensional glioma cell derivative (glioma cells, hydrogel, growth factors, proteins and medicines) is poured on the constructed vascular network pattern, so that nutrition supply of the glioma cell derivative can be realized, nutrition and oxygen can be provided for physiological, pathological and pharmacological models of the brain glioma cell three-dimensional derivative, and cell excreta can be taken away.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A preparation method of a brain glioma cell derivative applied to in vitro three-dimensional vascularization, which is characterized by comprising the following steps: the method comprises the following steps:
(1) Manufacturing a channel-patterned PDMS chip a;
(2) Manufacturing a PDMS film b, and thermally bonding the PDMS film b with the surface of the PDMS chip a where the channel pattern is located;
(3) Carrying out plasma treatment on one side of the chip with the film b, and bonding one side of the film b with a substrate c of which the surface is subjected to the plasma treatment to form a chip d;
(4) Filling hydrogel which is conducive to vascularization of endothelial cells into the channel of the chip d, placing the hydrogel into an incubator for dressing, and removing the hydrogel;
(5) Filling cells derived from cerebral microvessels and related growth factors into a channel of the chip d, placing the cells in an incubator, and peeling off the PDMS chip a after the cerebral microvessel cells are attached to the bottom surface of the channel;
(6) The culture solution is soaked in the cell pattern on the chip, and then placed in an incubator for culturing for a period of time, and the culture solution is removed to form a patterned multi-scale hierarchical vascular network;
(7) Casting a hydrogel body containing brain glioma cells, proteins, growth factors and medicines on the vascular network, wherein the vascular network can provide nutrition and oxygen for physiological, pathological and pharmacological models of the hydrogel body containing the brain glioma cells and take away cell excretions.
2. The method of manufacturing according to claim 1, wherein: the step (1) further comprises a step of performing hydrophilic treatment on the channel of the PDMS chip a.
Preferably, the hydrophilic treatment includes one or more of a combination of an ultraviolet ozone treatment, a polylysine coating, and a plasma treatment.
3. The method of manufacturing according to claim 1, wherein: the method also comprises the step of salinization treatment on the surface of the pattern of the PDMS chip a so as to reduce the adhesiveness of the PDMS chip a and the PDMS film b.
Preferably, the surface of the chip a pattern is subjected to salinization by adopting a chemical vapor deposition method.
It is further preferred to treat with fluorosilane or triphenylfluorosilicone.
4. The method of manufacturing according to claim 1, wherein: the method for manufacturing the PDMS film b in the step (2) adopts a spin coating method, the rotating speed of the spin coating machine is controlled to be 1000 rpm-3000 rpm, and the time is controlled to be 5-30 min.
Preferably, the thickness of the PDMS film b is 2 to 10 μm, preferably 4 to 6 μm, for example 5 μm.
5. The method of manufacturing according to claim 1, wherein: hydrogels that facilitate vascularization of endothelial cells include a mixed solution of one or more of fibronectin, fibrinogen, martigel;
for example, the hydrogel is a fibronectin solution with a concentration of 14-16 μg/mL.
6. The method of manufacturing according to claim 1, wherein: the gas used in the plasma treatment is N 2 、NH 3 、O 2 。
7. The method of manufacturing according to claim 1, wherein: in the step (4) and the step (5), the time of the culture in the incubator is 1-2 h.
Preferably, the brain microvascular cells are primary cells extracted from the human brain or cells of brain microvascular differentiated from ipscs; for example, brain microvascular endothelial cells, brain microvascular pericytes.
Preferably, the growth factor comprises a mixture of one or more of vEGF, EGF, FGF, hEGF and hFGF; for example, basic fibroblast growth factor and/or vascular endothelial growth factor.
8. The production method according to any one of claims 1 to 7, characterized in that: in the hydrogel body containing glioma cells, protein, growth factor and medicine, the density of glioma cells is 10 6 ～10 8 The elasticity model of the hydrogel is 100 kPa-1000 kPa.
Preferably, the protein is one or more of collagen, hyaluronic acid, brain tissue extracted extracellular matrix, neurotransmitter receptor protein, neuronal structural protein, synaptoprotein, nerve growth factor, ion channel protein, neuron specific enolone reductase, synaptosin, neuroinflammation and immune related protein, beta amyloid, alpha synuclein, ganglion protein, neuronal regulatory protein, neuronal conduction protein, motor protein and actin, neuroendocrine protein, brain derived lipoprotein, neuronal deoxyribonucleotide protein, zinc finger protein, neurotransmitter synthase, protein kinase C and protein kinase a.
Preferably, the agent is a mixture of one or more of an EGFR inhibitor, PI3K/AKT/mTOR signaling pathway inhibitor, angiogenesis inhibitor, chemotherapeutic agent, and immunotherapeutic agent.
The immunotherapeutic agent comprises one or more of an immune checkpoint inhibitor, CAR-T cell therapy, an immune vaccine, immune cell therapy, viral immunotherapy, and an immunostimulant agent.
9. A brain glioma cell derivative for in vitro three-dimensional vascularization, characterized in that: a method of preparing a brain glioma cell derivative according to any one of claims 1 to 8 for in vitro three-dimensional vascularization.
Preferably, the brain glioma cell derivative comprises a substrate, a patterned vascular network on the substrate, and a brain glioma covering the patterned vascular network.
10. Use of a brain glioma cell derivative according to claim 9 for in vitro three-dimensional vascularization in medical research, in particular in disease mechanism research, drug screening, therapeutic method development, radiation therapy research, vaccine research, gene editing and gene knockout research, tumor microenvironment research.
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