CN117535223A - Brain glioma cell derivative applied to in vitro three-dimensional vascularization as well as preparation method and application thereof - Google Patents

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

A kind of three-dimensional vascularized brain glioma cell derivative in vitro and its preparation Preparation methods and applications

Technical field

The invention belongs to the technical field of tissue engineering, and specifically relates to a three-dimensional vascularized brain glioma cell derivative in vitro and a preparation method thereof.

Background technique

Cell-cell and cell-microenvironment interactions play a crucial role in inducing functional expression of glioma cells. Natural tissues and organs are highly organized and possess complex vascular systems at multiple scales. The multi-scale vascular system can deliver nutrients and oxygen to surrounding cells and carry away cellular waste products. The construction of in vitro physiological, pathological, and pharmacological models requires engineering materials with good biocompatibility to simulate the natural extracellular matrix and microvascular system. Soft lithography, mold casting, bio-spinning, bio-3D printing, microfluidic patterning and other technologies have been used to develop 2D or 3D cell-containing structures. These micro-fabrication technologies can produce various multi-gradient, multi-material, multi-layer structures. style creature model. However, the construction of multi-scale complex vascularized networks within larger volumes of tissue is still challenging, sufficient nutrient supply cannot be achieved inside biological models, and guiding cells to grow for long periods of time in environmentally restricted spaces remains an urgent problem to be solved.

The current method used by 3D bioprinting technology in printing blood vessels is to accumulate cells layer by layer and then construct artificial tissue blood vessels through subsequent biological culture. The blood vessel cells produced by this method have low density and disorderly distribution, making it difficult to accurately control. At the same time, the blood vessel printing and molding method has problems such as slow molding speed, low efficiency, poor finished product structure, and inability to form multi-scale complex vascular systems.

Contents of the invention

In order to overcome the problems in the prior art, the present invention provides a three-dimensional vascularized brain glioma cell derivative in vitro and its preparation method and application. It adopts microfluidic patterning method and surface microstructure technology to produce a planar multi-dimensional The large-scale vascular network provides in vitro physiological, pathological, and pharmacological models with a brain glioma microenvironment that approximates the vascularization in vivo, and obtains a personalized tissue engineering vascular networked brain glioma biological model to better solve the problem. solves the problem of adequate vascularization within larger tissues.

The present invention is realized through the following technical solutions: a method for preparing brain glioma cell derivatives applied to three-dimensional vascularization in vitro, including the following steps:

(1) Make a channel-patterned PDMS chip a;

(2) Make a PDMS film b and thermally bond it to the surface of the PDMS chip a where the channel pattern is located;

(3) Plasma treatment is performed on the side of the chip with the film b, and then the side of the film b is bonded to the substrate c whose surface has been plasma treated to form chip d;

(4) Pour hydrogel that helps vascularize endothelial cells into the channel of chip d, then place it in an incubator and incubate it, and then remove the hydrogel;

(5) Pour cells derived from brain microvessels and related growth factors into the channel of chip d, place them in an incubator, and after the brain microvessel cells adhere to the bottom surface of the channel, peel off the PDMS chip a;

(6) Cover the cell pattern on the chip with the culture medium, then place it in an incubator for a period of time, then remove the culture medium to form a patterned multi-scale hierarchical vascular network;

(7) Pour a hydrogel containing brain glioma cells, proteins, growth factors and drugs on the vascular network to form a brain glioma cell derivative. The blood vessel network can be a hydrogel containing brain glioma cells. Physiological, pathological and pharmacological models of cellular hydrogels provide nutrients and oxygen and take away cellular excretions.

The present invention combines microfluidic pattern technology and surface microstructure manufacturing technology to create hierarchical and multi-scale protein, hydrogel, and cell patterns. At the same time, it can form micro-scale morphological features on the substrate and limit cells and guide directional growth of cells.

In one embodiment of the present invention, the step (1) also includes the step of hydrophilizing the channel of the PDMS chip a.

Preferably, the hydrophilic treatment includes but is not limited to one or more combinations of ultraviolet ozone treatment, polylysine coating, and N ₂ , NH ₃ , O ₂ gas plasma treatment, which can achieve the same hydrophilicity. Other treatments for water effects should also be included.

In one embodiment of the present invention, it also includes salting the surface of the PDMS chip a where the pattern is located to reduce the adhesion between the PDMS chip a and the PDMS film b.

Preferably, a chemical vapor deposition method is used to perform a salt treatment on the surface where the chip a pattern is located.

In chemical vapor deposition, the precursor gas is the starting material in the CVD process, and its selection depends on the required deposition material, which is usually an organic or inorganic compound, preferably fluorosilane or triphenylfluorosilane.

In one embodiment of the present invention, the method for making the PDMS film b in the step (2) is a spin coating method. The rotation speed of the spin coating machine is controlled to be 1000rpm~3000rpm and the time is 5~30min. Then, the PDMS film b and the PDMS The surface of chip A where the channel pattern is located is thermally bonded.

Preferably, the thickness of the PDMS film b is 2 to 10 μm, preferably 4 to 6 μm, such as 5 μm.

Preferably, the gases used in the plasma treatment are N ₂ , NH ₃ , and O ₂ .

Preferably, the substrate c is a glass sheet.

In one embodiment of the present invention, the hydrogel that facilitates endothelial cell vascularization includes one or more mixed solutions of fibronectin, fibrinogen, and martigel.

For example, the hydrogel is a fibronectin solution with a concentration of 14 to 16 μg/mL. The purpose of infusing fibronectin solution is to create a good microenvironment for the growth of brain microvascular cells. Any substance with the same effectiveness as fibronectin is optional.

Preferably, in the steps (4) and (5), the incubation time in the incubator is 1 to 2 hours.

In one embodiment of the present invention, the cells of the brain microvessels can be primary cells extracted from the human brain or cells of the brain microvessels differentiated from iPSCs. The cells of the brain microvessels include brain microvascular endothelial cells and brain microvascular pericytes. necessary cells.

In one embodiment of the present invention, the growth factor includes one or more mixtures of vEGF, EGF, FGF, hEGF and hFGF; for example, basic fibroblast growth factor and/or vascular endothelial cell growth factor.

In one embodiment of the present invention, in the hydrogel containing glioma cells, proteins, growth factors and drugs, the density of glioma cells is 10 ⁶ to 10 ⁸ cells/mL, and the elasticity of the hydrogel is The model is 100kPa～1000kPa.

In one embodiment of the present invention, the protein is collagen, hyaluronic acid, extracellular matrix extracted from brain tissue, neurotransmitter receptor protein (Neurotransmitter Receptor Proteins), neuron structural protein (Neuronal Structural Proteins), neurotransmitter Synaptic Proteins, Neurotrophic Factors, Ion Channel Proteins, neuron-specific enolone reductase (AKR1C1), synapsin, neuroinflammation and immune-related proteins (Neuroinflammatory andImmune-Related Proteins), β-amyloid (β-amyloid), α-synuclein (α-synuclein), neuroglobin, neuronal regulatory proteins (Neuronal Regulatory Proteins), neuronal transmission Neuronal Transport Proteins, Dynein and Actin, Neuronal Endocrine Proteins, Brain-derived Apolipoproteins, Neuronal DNA-binding Proteins, One or a mixture of one or more of Zinc Finger Proteins, Neurotransmitter Synthetic Enzymes, Protein Kinase C and Protein Kinase A.

In one embodiment of the present invention, the drugs are EGFR (epidermal growth factor receptor) inhibitors, PI3K/AKT/mTOR signaling pathway inhibitors, angiogenesis inhibitors, chemotherapy drugs, immunotherapy (including but not limited to immune examination One or more of point inhibitors, CAR-T cell therapy, immune vaccines, immune cell therapy, viral immunotherapy and immune stimulants) reagents.

The present invention also provides a brain glioma cell derivative applied to three-dimensional vascularization in vitro, which is obtained by using the above-mentioned preparation method of a brain glioma cell derivative.

In one embodiment of the present invention, the brain glioma cell derivative includes a base body, a patterned blood vessel network located on the base body, and a brain glioma covering the patterned blood vessel network.

The invention also provides an application of brain glioma cell derivatives for three-dimensional vascularization in vitro in medical research, especially in disease mechanism research, drug screening, treatment method development, radiotherapy research, vaccine research, Applications in gene editing and gene knockout research, and tumor microenvironment research.

Beneficial effects of the present invention:

1) The present invention provides a three-dimensional vascularized brain glioma cell derivative in vitro and a manufacturing method thereof, by producing a planar multi-scale vascular network (for example, specifically using microfluidic patterning method and surface microstructure technology) , and then pour a layer of three-dimensional glioma cell derivatives (glioma cells + hydrogel + growth factors + proteins + drugs) on the constructed blood vessel network pattern. The patterned multi-scale graded blood vessels can provide the above-mentioned nerve The physiological, pathological, and pharmacological models of the three-dimensional glioma cell derivatives provide nutrients and oxygen to realize the nutritional supply of the glioma cell derivatives and can also take away cell excretions. This method has good compatibility, simple production, low cost and strong universality.

2) This method is based on the principle of chip channel orientation and binding, simulating the microenvironment of vascularization in vivo, and obtaining glioma cell derivatives containing personalized tissue engineering vascular networks. The constructed multi-scale vascularization network can better solve the problem of sufficient vascularization within larger tissues, provide in vitro physiological, pathological, and pharmacological models with a glioma microenvironment that approximates in vivo vascularization, and obtain personalized The tissue-engineered vascular networked glioma biological model can be used in disease mechanism research, drug screening, treatment development, radiotherapy research, vaccine research, gene editing and gene knockout research, and tumor microenvironment research.

Description of drawings

Figure 1 is a manufacturing flow chart of PDMS chip a in Embodiment 1 of the present invention;

Figure 2 is a schematic diagram of the process of preparing a salt layer using chemical vapor deposition in Embodiment 1 of the present invention;

Figure 3 is a schematic structural diagram of the PDMS film b produced by spin coating in Embodiment 1 of the present invention;

Figure 4 is a schematic diagram of the bonding effect of PDMS chip a and PDMS film b in Embodiment 1 of the present invention;

Figure 5 is a schematic structural diagram of using particles such as oxygen to treat PDMS film b in Embodiment 1 of the present invention;

Figure 6 is a schematic structural diagram of the chip d after perfusion of brain microvascular cells in Embodiment 1 of the present invention;

Figure 7 is a schematic structural diagram of the PDMS chip a and chip d separated in Embodiment 1 of the present invention;

Figure 8 is a diagram of a multi-scale cerebral blood vessel network model formed on a glass substrate in Embodiment 1 of the present invention;

Figure 9 is a schematic diagram of the effect of pouring a layer of three-dimensional glioma cell derivatives on the cerebral blood vessel network pattern in Example 1 of the present invention.

Figure 10 is a schematic structural diagram of the mask used in Embodiment 1 of the present invention;

Figure 11 is a schematic diagram of the hierarchical multi-scale cerebral blood vessel network implemented by the present invention.

Detailed ways

The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following examples are only illustrative and explain the present invention, and should not be construed as limiting the scope of the present invention. All technologies implemented based on the above contents of the present invention are covered by the scope of protection intended by the present invention.

Example 1

(1) Using soft lithography technology to produce channel patterned PDMS chip a

Using wafers to prepare soft lithography processing templates:

Take the wafer, clean the surface, then set a layer of photoresist SU-8 on the wafer, and set a mask on the photoresist SU-8 (as shown in Figure 10, white is the light-transmitting part, black is the non-transparent part) Translucent part), dry at 95°C for 9 minutes. Turn on the nitrogen, set the exposure time to 18 seconds, use ultraviolet light for photolithography, and then dry at 95°C for 6 minutes. Use developer solution to develop, then rinse with ethanol, finally use nitrogen to dry the liquid, and then harden the film at 150°C for 30 minutes. The photoresist SU-8 used in the above steps is a negative photoresist, which is cured after being irradiated with ultraviolet light. The uncured photoresist is washed away with the SU-8 developer and then dried.

Use photoresist SU8-2100 to remove the glue again (same as above), and then dry it at 95°C for 1 hour. After aligning the upper and lower layers, set the exposure time to 20 seconds, bake at 95°C for 10 minutes, use a developer again to develop, and then harden the film at 150°C-180°C for 30 minutes to obtain a soft photolithography processing template.

PDMS chip a preparation and oxygen plasma treatment steps:

1. Place the soft photolithography processing template prepared above into a sealed petri dish with a diameter of 100 mm, and then add trimethylchlorosilane into the container. After the trimethylchlorosilane evaporates, the surface of the template will be hydrophobically treated.

2. Mix polydimethylsiloxane (PDMS) glue A and glue B (brand: Dow Corning, model: SYLGARD 184) in a mass ratio of 10:1-5:1 to obtain a PDMS glue with a total weight of 15-20g .

3. Pour the PDMS glue on the above-mentioned hydrophobically treated soft photolithography template, place it in a vacuum drying box, and remove air bubbles.

4. Place in the oven and bake at 80°C for 0.5-1h.

5. After cooling to normal temperature, remove the patterned PDMS from the template to obtain PDMS chip a.

(2) The surface of PDMS chip a is hydrophobic. One side of the channel of chip a is treated with oxygen plasma for 1 minute at a power of 100~500W. Oxygen plasma treatment will increase the adhesion ability of the patterned PDMS structure surface and make it hydrophilic. sexual characteristics.

(3) As shown in Figure 2, chemical vapor deposition is used to process one side of the chip channel to salt the surface, making the PDMS chip a easy to peel off in step (8).

(4) As shown in Figure 3, use the spin coating method to make the PDMS film b. Control the rotation speed of the spin coater to 1000rpm to 3000rpm and rotate for 5 to 30 minutes so that the thickness of the PDMS film b is about 5 μm. Then, add the PDMS film b and The surface of the PDMS chip a where the channel pattern is located is thermally bonded to obtain the structure shown in Figure 4.

(5) Use oxygen plasma to perform plasma treatment on the other side of the PDMS film b (power 100~500W, treatment time 1 min).

(6) Bond the chip film b and the glass sheet c whose surface has been plasma treated to form chip d, as shown in Figure 6. Pour 15 μg/mL fibronectin solution into channel d of the chip, incubate it in an incubator for 2 hours, and then use sterile gauze, dust-free absorbent paper, pipette, etc. to remove the fibrin solution.

(7) Continue to pour human brain microvascular cells, human brain vascular pericytes and cell growth factors (vEGF, FGF, EGF) into the channel of chip d, place them in an incubator and culture them until the brain microvascular cells adhere to the bottom of the channel. After surface treatment, peel off the PDMS chip a after about 2 hours, as shown in Figure 7.

(8) Submerge the cell pattern on the chip (after peeling off the PDMS chip a) with the culture medium, and then place it in an incubator for culture to form a patterned tissue engineering vascular network, as shown in Figures 8 and 11.

(9) After continuing to culture the vascular network for 3 days, remove the culture medium and pour on the vascular network a solution containing brain glioma cells (10 ⁶ ~ 10 ⁸ cells/mL), proteins, and growth factors (such as vEGF (1ng/mL) ~50ng/mL), EGF (1ng/mL~100ng/mL), FGF (1ng/mL~100ng/mL)), drug hydrogel (elastic modulus 100kPa~1000kPa), and many patterned gradations can be obtained Scale cerebrovascular network, as shown in Figure 9.

Figure 11 shows the hierarchical multi-scale cerebral vascular network of PDMS chip a with channel widths of 300 μm, 200 μm, 150 μm and 75 μm.

The present invention can realize the derivation of glioma cells by pouring a layer of three-dimensional glioma cell derivatives (glioma cells + hydrogel + growth factor + protein + drug) on the constructed blood vessel network pattern. The body's nutrient supply can provide nutrients and oxygen to the physiological, pathological and pharmacological models of three-dimensional derivatives of brain glioma cells, and take away cell excretions.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above-described embodiment. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall 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.