CN114369532B - Microfluidic cell culture device simulating filtration bleb and application thereof - Google Patents
Microfluidic cell culture device simulating filtration bleb and application thereof Download PDFInfo
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Abstract
The invention provides a microfluidic cell culture device for simulating a filtration bubble and application thereof, wherein the microfluidic cell culture device for simulating the filtration bubble comprises a cover plate, a cell culture main body and a grooved bottom plate containing a micro channel from top to bottom; the cell culture main body comprises a cell culture cavity with a flat bottom and a semi-arc dome-shaped bulge; and the grooved bottom plate is carved with an inflow channel, an outflow channel and a summary outflow channel. The invention also provides a use method of the microfluidic cell culture device for simulating the filtration bleb. The microfluidic cell culture device for simulating the filter bleb simulates the biological form of the filter bleb and the unidirectional and dynamic flowing process of aqueous humor in eyes, can culture cells in a state similar to natural physiology and study functions of the cells, and has important significance for the study of related diseases.
Description
Technical Field
The invention belongs to the technical field of cell in-vitro culture devices, and particularly relates to a microfluidic cell culture device simulating a filtration membrane and application thereof.
Background
Glaucoma is the first irreversible blinding eye disease in the global incidence, the pathogenesis of which is manifested as damage to the ocular nerve by pathological ocular hypertension. Trabeculectomy has been widely used for the treatment of glaucoma since the 60 s of the 20 th century as an effective means of lowering intraocular pressure and as a gold standard for anti-glaucoma surgical treatment.
Trabeculectomy aims to properly drain aqueous humor from the eye to the scleral sump by resecting portions of trabecular meshwork tissue, thereby reducing pathologically high intraocular pressure, and eventually aqueous humor accumulates between fascia tissue and scleral tissue to form a bleb. Aqueous humor in the filter bleb flows back to the whole body via the vascular system and lymphatic system. The bleb morphology after trabeculectomy is shown in figure 1. Wherein, the a picture is a filtering bleb picture obtained by shooting (Canon) of the anterior ocular segment, and can be seen to be positioned above the cornea to form a bleb bulge structure; figure b is a cross-sectional view of a bleb obtained by optical coherence tomography (Optovue, 6 x 6-mm HD Angio Retina protocol) of the anterior ocular segment in the form of a raised approximately semicircular structure.
Blebs formed after trabeculectomy are the primary space to contain excess aqueous humor within the eye, and scarring is the leading cause of bleb loss of function, leading to surgical failure. Therefore, the study of maintenance of the function of the postoperative bleb has been an important task for glaucoma doctors.
Currently, research in this field is focused mainly on the in vivo level by constructing animal models of blebs formed by trabeculectomy. At the cellular level, selection of 2D culture also hardly reflects the function of blebs, and simulation of blebs is difficult to achieve. The aqueous humor reaches the filtering bleb from the eye and then enters the circulating system, which is a dynamic process, and the conventional static in-vitro 2D cell culture cannot simulate the fluid field in the stereoscopic filtering bleb and cannot realize the microfluidic circulation of the culture solution.
Pressure and extracellular matrix rigidity are important factors affecting cell function. With the fluctuation of intraocular pressure after operation, the functions of cells inside and outside the eyes are affected to different degrees. Tissue engineering has been focusing on the effect of extracellular matrix stiffness on cell function over the last decade, involving very complex mechanisms. On the one hand, the extracellular matrix can provide chemical and mechanical signals to influence the cell behaviors, and the cells convert the signals into chemical stimuli through different sensors so as to regulate the cell functions, so that the signals are key factors for repairing, reconstructing and healing wound tissues; on the other hand, changes in the mechanical properties exhibited by diseased tissue can lead to impairment of normal cellular function. As bleb scarring progresses, scar tissue is closely related to stiffness, and there must be a significant interaction between cellular function and extracellular matrix stiffness.
Therefore, how to provide a cell culture device capable of dynamically simulating the function of a filter membrane and regulating the hardness of extracellular matrix has become a problem to be solved.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides a microfluidic cell culture device for simulating a filter bubble and application thereof, which can simulate the dynamic environment that aqueous humor enters the filter bubble from the eye and flows back into a vascular lymphatic vessel, and can explore the influence of different culture conditions on the functions and physiological states of cells by adjusting the cell culture conditions and extracellular matrix hardness.
To achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a microfluidic cell culture device simulating a filtration pore, wherein the microfluidic cell culture device simulating the filtration pore comprises a cover plate, a cell culture main body and a grooved bottom plate containing a microchannel from top to bottom;
the cell culture main body comprises a cell culture cavity with a flat semi-arc dome-shaped bulge at the bottom, the cell culture cavity is used for accommodating cells, hydrogel and perfusate, the hardness of different extracellular matrixes is simulated by changing the hardness of the hydrogel, and the fluctuation of intraocular pressure is simulated by adjusting the perfusion pressure;
the cell culture device comprises a cell culture cavity, and is characterized in that an inflow channel, an outflow channel and a summary outflow channel are engraved on the engraved bottom plate, the inflow channel is connected to the short arc edge of the cell culture cavity, the outflow channel is connected to the long arc edge of the cell culture cavity, the number of the outflow channels is multiple, the short arc edge of the cell culture cavity simulates cornice limbus, and the long arc edge of the cell culture cavity simulates filtering blebs and is along an arc boundary.
According to the microfluidic cell culture device simulating the filter bleb, a set of dynamic culture system with variable pressure and variable hardness is provided for cells based on a microfluidic system, the culture system enters an inflow channel from a liquid injection hole, reaches a cell culture cavity, flows out from a plurality of outflow channels finally at a liquid discharge hole to form a dynamic unidirectional flow system, simulates a dynamic environment that aqueous humor enters the filter bleb from the inside of an eye after trabeculectomy and flows back into a vascular lymphatic vessel, and detects cell functions under different intraocular pressures and different extracellular matrix hardness conditions.
In the invention, the cell culture cavity is a dome-shaped semicircular device, the short arc line edge simulates a cornice sclera edge, the long arc line edge simulates the arc boundary of the outer edge of the filtering bleb, the shape is similar to the physiological structure of the filtering bleb after trabeculectomy, the fluid field of aqueous humor inside the filtering bleb is simulated to a certain extent, and cells growing in the cell culture cavity with the shape are regulated and controlled by more biomechanical parameters and are more approximate to the physiological state in vivo.
Under normal physiological conditions, aqueous humor is produced by the ciliary body in the form of active transport, ultrafiltration and dispersion, at a rate of about 1.5 to 3 μl/min. The aqueous humor contains lactic acid, vitamin C, glucose, inositol, glutathione, urea, sodium, potassium, chlorine and other ions, and some growth regulating factors, such as TGF-b, aFGF, bFGF, etc. The oxygen partial pressure of the aqueous humor is about 55 mmHg, the carbon dioxide partial pressure is 40-60 mmHg, and the outflow coefficient is 0.22-0.28 mu L/(min multiplied by mmHg). According to the microfluidic cell culture device based on the simulated filtration bleb, not only can the aqueous humor velocity under physiological conditions be reproduced, but also the aqueous humor microenvironment under pathological conditions can be quickly and conveniently constructed by changing the components of the perfusate. Meanwhile, the normal intraocular pressure range is 10-21 mmHg, and the normal intraocular pressure microenvironment and the pathological ocular hypertension microenvironment can be simulated by adjusting the perfusion pressure, so that the change of cell functions under the action of different pressures is observed.
In the present invention, the inflow channel simulates a trabecular meshwork incision, i.e., the passage of aqueous humor from within the eye to the bleb.
In the invention, the outflow channel simulates a return passage of aqueous humor in the filtering bleb through subconjunctival vascular lymphatic vessels, and the aqueous humor flows back into the blood vessels and lymphatic vessels from the filtering bleb under a natural physiological state.
Preferably, the number of the inflow channels is at least 1, for example, 1, 2, 3, 4 or 5, and other specific point values in the numerical range may be selected, which will not be described in detail herein.
Preferably, the number of the outflow channels is at least 3, for example, 3, 4, 5, 6, 7, 8, 9 or 10, and other specific values in the numerical range may be selected, which will not be described in detail herein.
Preferably, the number of the summary outflow channels is 1.
Preferably, the inflow channel has a rectangular cross section.
Preferably, the length of the long side of the rectangle is 7-10 mm, for example, it may be 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm or 10 mm, etc., and the length of the short side is 1-3 mm, for example, it may be 1 mm, 1.5 mm, 2 mm, 2.5 mm or 3 mm, etc., and other specific point values within the numerical range are all selectable and will not be described herein again.
Preferably, the outflow channel is circular in cross section.
Preferably, the diameter of the circle is 180-230 μm, for example, it may be 180 mm, 190 mm, 200 mm, 210 mm, 220 mm or 230 mm, and other specific values within the numerical range may be selected, which will not be described herein.
Preferably, the top of the cell culture chamber is provided with a glue injection hole.
In the invention, the injecting holes are used for injecting hydrogel precursors and exhausting air.
Preferably, the cell culture main body is further provided with a liquid injection hole and a liquid discharge hole.
Preferably, the liquid injection hole is connected with a perfusion tube, and the perfusion tube is connected with a constant-speed injector.
Preferably, the drain hole is connected to a drain pipe.
Preferably, the preparation material of the cell culture body comprises silica gel.
In the invention, silica gel is selected as a preparation material of a cell culture main body, and can transmit ultraviolet light, so that hydrogel is convenient to gel; the silica gel has certain mechanical hardness and is adjustable in a certain range, so that the normal tissue hardness can be simulated; the silica gel has better permeability, and is convenient for observing the growth condition of cells in the cell culture cavity.
Preferably, the cover plate is made of PMMA.
In the invention, PMMA is selected as the preparation material of the cover plate, the light transmittance is good, the radiation of ultraviolet light to the hydrogel is not influenced, and meanwhile, the cover plate has good mechanical property, weather resistance and formability, and the cover plate has good sealing effect on the device.
As a preferable technical scheme, the microfluidic cell culture device for simulating the filtration bubble comprises a cover plate, a cell culture main body and a grooved bottom plate containing a microchannel from top to bottom;
the cell culture main body comprises a cell culture cavity with a flat bottom and a semi-arc dome-shaped bulge;
an inflow channel, an outflow channel and a summary outflow channel are engraved on the grooved bottom plate;
the number of the inflow channels is at least 1, and the inflow channels are connected with the short arc line side of the cell culture cavity;
the cross section of the inflow channel is rectangular, the length of the long side of the rectangle is 7-10 mm, and the length of the short side of the rectangle is 1-3 mm;
the number of the outflow channels is at least 3, and the outflow channels are connected to the long arc line side of the cell culture cavity;
the cross section of the outflow channel is circular, and the diameter of the circular shape is 180-230 mu m;
the number of the summarized outflow channels is 1;
the top of the cell culture cavity is provided with a glue injection hole;
the cell culture main body is also provided with a liquid injection hole and a liquid discharge hole, the liquid injection hole is connected with a perfusion tube, the perfusion tube is connected with a constant-speed injector, and the liquid discharge hole is connected with a liquid discharge tube;
the preparation material of the cell culture main body comprises silica gel, and the preparation material of the cover plate comprises PMMA.
In a second aspect, the present invention provides a method for using the microfluidic cell culture device simulating a filtration pore according to the first aspect, the method comprising:
mixing the cells with the hydrogel, and injecting the mixture into a cell culture cavity through a glue injection hole;
covering a cover plate and a fixing device;
regulating the flow rate of the constant-speed injector, injecting the perfusion liquid from the perfusion tube, collecting and discharging the waste liquid in the liquid discharge tube, and simulating the fluctuation of intraocular pressure by regulating the perfusion pressure;
culturing the cells and observing;
the hydrogel comprises 5% -15% of gelatin by mass, and the change of extracellular matrix and tissue hardness in the process of scar formation of the filtration bleb is simulated by adjusting the hardness of the hydrogel.
Preferably, the hydrogel includes gelatin, the mass fraction of the gelatin is 5% -15%, for example, may be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%, etc., and other specific point values in the numerical range may be selected, which will not be described in detail herein.
In the invention, the hydrogel component mainly comprises collagen I and collagen III, and a small amount of collagen IV, V, VI, VIII, XII and XIII, proteoglycan and non-collagen glycoprotein, which are close to biological components of sclera, so that the extracellular matrix microenvironment between scleral surface and Tenon capsule tissue can be well simulated. In addition, by changing the hardness of the hydrogel, the hardness of different extracellular matrixes can be simulated, which is beneficial to researching the external regulating factors of the cell functions in the ineffective filtering bleb forming process.
Preferably, the density of the cells in the hydrogel is (1-5) ×10 2 mu.L, for example, may be 1X 10 2 mu.L, 2X 10 2 mu.L, 3X 10 2 mu.L, 4X 10 2 mu.L or 5X 10 2 And other specific point values within the numerical range can be selected, and the details are not repeated here.
Preferably, the flow rate of the constant-speed injector is 0.5-2 mL/day, for example, may be 0.5 mL/day, 1 mL/day, 1.5 mL/day, or 2 mL/day, and other specific point values within the numerical range may be selected, which will not be described in detail herein.
In the present invention, the culture conditions of the cells may be determined according to the type of the cells to be cultured.
As a preferable technical scheme, the application method of the microfluidic cell culture device simulating the filtration pore comprises the following steps:
mixing the cells with 5-15% gelatin, wherein the density of the mixed cells in the gelatin is (1-5) multiplied by 10 2 Mu L, injecting into the cell culture cavity through the glue injection hole;
covering the cover plate after the glue is formed, and fixing all parts of the device;
regulating the flow rate of the constant-speed injector to 0.5-2 mL/day, injecting the perfusion liquid from the perfusion tube, collecting the waste liquid in the liquid discharge tube and discharging;
the culture conditions were determined, and the cells were cultured and observed.
In a third aspect, the present invention provides an application of the microfluidic cell culture device simulating a filtration pore according to the first aspect and/or the use method of the microfluidic cell culture device simulating a filtration pore according to the second aspect in preparing a cell dynamic model under the condition of simulating a filtration pore.
In a fourth aspect, the invention provides a cell dynamic model under the condition of a simulated filtration membrane, which is obtained by culturing the microfluidic cell culture device of the simulated filtration membrane in the first aspect and/or the microfluidic cell culture device of the simulated filtration membrane in the second aspect.
Compared with the prior art, the invention has the following beneficial effects:
(1) In the invention, the microfluidic cell culture device simulating the filtration bleb simulates the filtration bleb formed after glaucoma trabeculectomy, wherein an inflow channel simulates trabecular tissue incision, and a plurality of outflow channels simulate reflux blood vessels and lymphatic vessels; the shape of the cell culture cavity refers to the semi-arc dome-shaped bulge structure of the bleb, and is more in accordance with the biological shape of the bleb;
(2) The invention is based on a microfluidic system, cell culture solution is injected in one way from an inflow channel and finally flows out from an outflow channel, so that a one-way microfluidic system of the inflow channel, a cell culture cavity and the outflow channel is constructed, and the microfluidic system is consistent with the flow direction of aqueous humor flowing from an eye to a filtering bulb through a trabecular meshwork incision and then flowing back through a vascular lymphatic vessel; the flow rate of the microfluidic liquid is controllable, so that the flow rate of the aqueous humor in the filtering bleb can be simulated to slowly flow out of the eye, the aqueous humor is dispersed into the dynamic process of blood circulation, and the fluctuation of the intraocular pressure can be simulated by adjusting the perfusion pressure;
(3) The invention adopts the 3D cell culture assisted by hydrogel, the hydrogel component is close to the biological component of human sclera, and provides a microenvironment which is more close to the living condition in vivo for cell culture; by adjusting the hardness of the hydrogel, the change of extracellular matrix and tissue hardness in the process of scar formation of the filtering bleb is simulated, so that the influence of the extracellular matrix hardness on the cell function is favorably researched; the 3D microfluidic cell culture system has the advantages of dynamic, variable pressure, variable hardness and the like which are incomparable with the traditional 2D cell culture, is closer to the shape of the filtration bleb in a physiological state, is favorable for researching cell functions and regulation of the filtration bleb, can dynamically control and observe the physiological and pathological processes in real time, and improves the research level of diseases and the research and development efficiency of medicines.
Drawings
Fig. 1 is a morphological picture of a bleb after trabeculectomy, wherein a is a bleb picture obtained by photographing an anterior segment of an eye, and b is a bleb cross-sectional picture obtained by optical coherence tomography of the anterior segment of an eye;
FIG. 2 is a central vertical cross-section of a microfluidic cell culture device simulating a filter bulb with glue injection holes parallel to the long sides;
FIG. 3 is a horizontal cross-sectional view of a microfluidic cell culture device simulating a filter bleb;
(in the figure, 1-cover plate, 2-cell culture main body, 3-grooved bottom plate, 4-glue injection hole, 5-liquid injection hole, 6-inflow channel, 7-cell culture cavity, 8-outflow channel, 9-liquid discharge hole and 10-summary outflow channel)
FIG. 4 is a microscopic photograph of cultured fibroblasts, wherein, a is a photograph of cell spheres formed after 3D hydrogel culture using a microfluidic cell culture device simulating a filter bulb in example 4, and b is a photograph of cells in comparative example 1 which were subjected to an adherent culture by a conventional 2D culture method (magnification: 10×);
FIG. 5 is a graph showing the absorbance detection results of fibroblasts cultured in examples 4 to 6;
fig. 6 is a graph showing PI staining results of fibroblasts cultured in examples 4 to 6 (scale bar=100 μm).
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1
The embodiment provides a microfluidic cell culture device simulating a filter bubble, which is shown in fig. 2 as a vertical cross-sectional view of the center of a glue injection hole 4 parallel to a long side, and is shown in fig. 3 as a horizontal cross-sectional view.
The microfluidic cell culture device for simulating the filtration bleb comprises a cover plate 1, a cell culture main body 2 and a grooved bottom plate 3 containing a microchannel from top to bottom;
the cell culture main body 2 comprises a cell culture cavity 7 with a flat semi-arc dome-shaped bulge at the bottom;
an inflow channel 6, an outflow channel 8 and a summary outflow channel 10 are engraved on the grooved bottom plate 3;
the number of the inflow channels 6 is 1, and the inflow channels are connected with the short arc edges of the cell culture cavity 7;
the cross section of the inflow channel 6 is rectangular, the length of the long side of the rectangle is 8 mm, and the length of the short side is 2 mm;
the number of the outflow channels 8 is 11, and the outflow channels are connected with the long arc sides of the cell culture cavity 7;
the cross section of the outflow channel 8 is circular, and the diameter of the circular shape is 220 μm;
the number of the summarized outflow channels 10 is 1;
the top of the cell culture cavity 7 is provided with a glue injection hole 4;
the cell culture main body 2 is also provided with a liquid injection hole 5 and a liquid discharge hole 9, the liquid injection hole 5 is connected with a perfusion tube, the perfusion tube is connected with a constant-speed injector, and the liquid discharge hole 9 is connected with a liquid discharge tube;
the preparation material of the cell culture main body 2 is silica gel, and the preparation material of the cover plate 1 is PMMA.
Example 2
The embodiment provides a microfluidic cell culture device simulating a filter bubble, which is shown in fig. 2 as a vertical cross-sectional view of the center of a glue injection hole 4 parallel to a long side, and is shown in fig. 3 as a horizontal cross-sectional view.
The microfluidic cell culture device for simulating the filtration bleb comprises a cover plate 1, a cell culture main body 2 and a grooved bottom plate 3 containing a microchannel from top to bottom;
the cell culture main body 2 comprises a cell culture cavity 7 with a flat semi-arc dome-shaped bulge at the bottom;
an inflow channel 6, an outflow channel 8 and a summary outflow channel 10 are engraved on the grooved bottom plate 3;
the number of the inflow channels 6 is 1, and the inflow channels are connected with the short arc edges of the cell culture cavity 7;
the cross section of the inflow channel 6 is rectangular, the length of the long side of the rectangle is 7 mm, and the length of the short side is 3 mm;
the number of the outflow channels 8 is 11, and the outflow channels are connected with the long arc sides of the cell culture cavity 7;
the cross section of the outflow channel 8 is circular, and the diameter of the circular shape is 230 μm;
the number of the summarized outflow channels 10 is 1;
the top of the cell culture cavity 7 is provided with a glue injection hole 4;
the cell culture main body 2 is also provided with a liquid injection hole 5 and a liquid discharge hole 9, the liquid injection hole 5 is connected with a perfusion tube, the perfusion tube is connected with a constant-speed injector, and the liquid discharge hole 9 is connected with a liquid discharge tube;
the preparation material of the cell culture main body 2 is silica gel, and the preparation material of the cover plate 1 is PMMA.
Example 3
The embodiment provides a microfluidic cell culture device simulating a filter bubble, which is shown in fig. 2 as a vertical cross-sectional view of the center of a glue injection hole 4 parallel to a long side, and is shown in fig. 3 as a horizontal cross-sectional view.
The microfluidic cell culture device for simulating the filtration bleb comprises a cover plate 1, a cell culture main body 2 and a grooved bottom plate 3 containing a microchannel from top to bottom;
the cell culture main body 2 comprises a cell culture cavity 7 with a flat semi-arc dome-shaped bulge at the bottom;
an inflow channel 6, an outflow channel 8 and a summary outflow channel 10 are engraved on the grooved bottom plate 3;
the number of the inflow channels 6 is 1, and the inflow channels are connected with the short arc edges of the cell culture cavity 7;
the cross section of the inflow channel 6 is rectangular, the length of the long side of the rectangle is 7 mm, and the length of the short side is 1 mm;
the number of the outflow channels 8 is 11, and the outflow channels are connected with the long arc sides of the cell culture cavity 7;
the cross section of the outflow channel 8 is circular, and the diameter of the circular shape is 180 μm;
the number of the summarized outflow channels 10 is 1;
the top of the cell culture cavity 7 is provided with a glue injection hole 4;
the cell culture main body 2 is also provided with a liquid injection hole 5 and a liquid discharge hole 9, the liquid injection hole 5 is connected with a perfusion tube, the perfusion tube is connected with a constant-speed injector, and the liquid discharge hole 9 is connected with a liquid discharge tube;
the preparation material of the cell culture main body 2 is silica gel, and the preparation material of the cover plate 1 is PMMA.
Example 4
In this example, the microfluidic cell culture apparatus simulating the filtration pore in example 1 was used to culture fibroblasts as follows:
fibroblasts (primary human Tenon cyst fibroblasts, preparation method see Yin X, sun H, yu D, liang Y, yuan Z, ge Y Hydroxycamptothecin induces apoptosis of human Tenon's capsule fibroblasts by activating the PERK signaling path. Invest Ophthalmol Vis Sci.2013, 54 (7): 4749-4758) were dissolved with 10% gelatin (5 g gel (available from Henan Brilliant Biotechnology Co., ltd.) in 50 mL PBS, stirred at 50℃until dissolution, 6 mL methacrylic anhydride was added dropwise while adjusting pH to 7.5-8 with 5M NaOH, after reaction 4H, and lyophilized to give gelatin dry powder for use, gelatin dry powder was weighed in the experiment, PBS and 1% LAP photoinitiator (available from Guangxi biological medical materials Co., ltd.) were added to prepare 10% liquid gelatin) were mixed, and the density of the mixed cells in gelatin was 2X 10% 2 mu.L of gelatin was injected into the cell culture chamber 7 through the glue injection hole 4 at a concentration of 100. Mu.L;
ultraviolet irradiation to gel gelatin, covering the cover plate 1 and fixing the parts of the device;
the flow rate of the constant-speed injector was adjusted to 1 mL/day, a perfusion solution (DMEM (high sugar medium available from Gibco) containing 5% fetal bovine serum (available from Gibco)) was injected from a perfusion tube, and the waste liquid in the drain tube was collected and discharged;
at 37℃with 5% CO 2 In the cell incubator, cells were cultured and observed.
Example 5
In this example, the microfluidic cell culture apparatus simulating the filtration pore in example 1 was used to culture fibroblasts as follows:
mixing fibroblast with 15% gelatin, and mixing to obtain a density of 2×10 2 mu.L of gelatin was injected into the cell culture chamber 7 through the glue injection hole 4 at a concentration of 100. Mu.L;
ultraviolet irradiation to gel gelatin, covering the cover plate 1 and fixing the parts of the device;
regulating the flow rate of the constant-speed injector to be 0.5 mL/day, injecting the perfusion liquid from the perfusion tube, collecting the waste liquid in the liquid discharge tube and discharging;
at 37℃with 5% CO 2 In the cell incubator, cells were cultured and observed.
The preparation method of gelatin and fibroblasts was the same as in example 4.
Example 6
In this example, the microfluidic cell culture apparatus simulating the filtration pore in example 1 was used to culture fibroblasts as follows:
mixing fibroblast with gelatin with mass fraction of 5%, and mixing to obtain cell density of 2X10 in gelatin 2 mu.L of gelatin was injected into the cell culture chamber 7 through the glue injection hole 4 at a concentration of 100. Mu.L;
ultraviolet irradiation to gel gelatin, covering the cover plate 1 and fixing the parts of the device;
regulating the flow rate of the constant-speed injector to be 2 mL/day, injecting the perfusion liquid from the perfusion tube, collecting the waste liquid in the liquid discharge tube and discharging the waste liquid;
at 37℃with 5% CO 2 In the cell incubator, cells were cultured and observed.
The preparation method of gelatin and fibroblasts was the same as in example 4.
Example 7
In this example, the microfluidic cell culture apparatus simulating the filtration pore in example 2 was used to culture fibroblasts, and the steps were as follows:
mixing fibroblast with 15% gelatin, and mixing to obtain a density of 1×10 2 mu.L of gelatin was injected into the cell culture chamber 7 through the glue injection hole 4 at a concentration of 100. Mu.L;
ultraviolet irradiation to gel gelatin, covering the cover plate 1 and fixing the parts of the device;
regulating the flow rate of the constant-speed injector to be 0.5 mL/day, injecting the perfusion liquid from the perfusion tube, collecting the waste liquid in the liquid discharge tube and discharging;
at 37℃with 5% CO 2 In the cell incubator, cells were cultured and observed.
The preparation method of gelatin and fibroblasts was the same as in example 4.
Example 8
In this example, the microfluidic cell culture apparatus simulating the filtration pore in example 3 was used to culture fibroblasts, and the steps were as follows:
mixing fibroblast with gelatin with mass fraction of 5%, and mixing to obtain cell density of 5×10 in gelatin 2 mu.L of gelatin was injected into the cell culture chamber 7 through the glue injection hole 4 at a concentration of 100. Mu.L;
ultraviolet irradiation to gel gelatin, covering the cover plate 1 and fixing the parts of the device;
regulating the flow rate of the constant-speed injector to be 2 mL/day, injecting the perfusion liquid from the perfusion tube, collecting the waste liquid in the liquid discharge tube and discharging the waste liquid;
at 37℃with 5% CO 2 In the cell incubator, cells were cultured and observed.
The preparation method of gelatin and fibroblasts was the same as in example 4.
Comparative example 1
The comparative example carried out a conventional culture of fibroblasts, the steps being as follows:
uniformly inoculating logarithmic phase fibroblast into 96-well plate, about 5000 cells per well, and culturing at 37deg.C and 5% CO 2 After culturing 24 h in a cell culture incubator, observations and experiments were performed.
The method for preparing fibroblasts was the same as in example 4.
Morphological observation
The microscopic picture of the fibroblasts cultured in example 4 is shown as a graph in fig. 4, and the microscopic picture of the fibroblasts cultured in comparative example 1 is shown as b graph in fig. 4.
As can be seen from fig. 4, the fibroblast cultured by using the microfluidic cell culture device simulating the filtration bleb in the invention has a spherical shape, clear morphology and less content, and has a great difference from the morphology of the fibroblast cultured by the conventional method. In addition, the ability of cells to migrate, adhere, proliferate, etc. under 2D and 3D culture conditions is also essentially different.
The morphology of the fibroblasts cultured in examples 5-8 is similar to that of example 4, and for the sake of brevity, description thereof will be omitted.
Cell activity assay
The fibroblast cells cultured in examples 4 to 6 were tested for cell activity, and the effect of different gelatin concentrations on the physiological state of the cells was tested.
MTT assay
The method of examples 4 to 6 was used to culture fibroblasts, and after 24 to h culture, cytotoxicity was detected by MTT method, and the steps were as follows:
taking out the hydrogel in the device, placing the hydrogel in a 96-well plate, adding 100 mu L of DMEM culture medium without serum into each well, adding 20 mu L of MTT solution (purchased from Biyun biotechnology Co., ltd.) with the concentration of 5 mg/mL, and continuously culturing in an incubator at 37 ℃ for 4 h;
stopping culturing, carefully sucking out the culture solution in the holes, adding 150 μl of DMSO into each hole, and shaking at low speed for 10 min to dissolve completely;
the absorbance (a value) of each well was measured at 490 nm wavelength using an enzyme-linked immunosorbent assay, the mean value of 3 wells per group was taken and the results were counted.
The absorbance detection results of the cells are shown in FIG. 5. As can be seen, there is a clear difference in absorbance of the three groups of cells (p < 0.05), indicating that the hardness of the extracellular matrix has an effect on the physiological state of the cells, the cell proliferation capacity in the soft matrix group is significantly higher than in the medium and hard matrix groups.
PI staining method detection
The method of examples 4 to 6 was used to culture fibroblasts, and after 24 to h culture, the number of dead cells was detected by PI staining, as follows:
the hydrogel in the device was removed, placed in 96-well plates, 100. Mu.L of serum-free DMEM medium was added to each well, and 1. Mu.L of PI staining solution was added thereto, incubated at 4℃for 30 min in the absence of light, and observed.
The PI staining results of the cells are shown in FIG. 6. As can be seen from the graph, the fluorescence intensity of the cells of example 5 group was more pronounced than that of example 4 group and example 6 group, indicating that the hardness of gelatin was related to the survival state of the cells, and the greater the hardness of the extracellular matrix, the greater the number of dead cells.
In summary, the invention provides a microfluidic cell culture device for simulating a filter bubble, the structure of the microfluidic cell culture device is very similar to the biological form of the filter bubble, and the microfluidic cell culture device simulates the unidirectional dynamic flow of aqueous humor flowing back into a vascular lymphatic vessel from the filter bubble in eyes, so that a dynamic culture system with variable pressure and hardness is provided for cells, the physiological state of the eyes after trabeculectomy is simulated, the functions of the cells in the environment are further researched, and tools are provided for research of related diseases and research and development of medicines.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (19)
1. The microfluidic cell culture device simulating the filtration bleb is characterized by comprising a cover plate, a cell culture main body and a grooved bottom plate containing a microchannel from top to bottom;
the cell culture main body comprises a cell culture cavity with a flat semi-arc dome-shaped bulge at the bottom, the cell culture cavity is used for accommodating cells, hydrogel and perfusate, the hardness of different extracellular matrixes is simulated by changing the hardness of the hydrogel, and the fluctuation of intraocular pressure is simulated by adjusting the perfusion pressure;
the cell culture device comprises a cell culture cavity, and is characterized in that an inflow channel, an outflow channel and a summary outflow channel are engraved on the engraved bottom plate, the inflow channel is connected to the short arc edge of the cell culture cavity, the outflow channel is connected to the long arc edge of the cell culture cavity, the number of the outflow channels is multiple, the short arc edge of the cell culture cavity simulates cornice limbus, and the long arc edge of the cell culture cavity simulates filtering blebs and is along an arc boundary.
2. The microfluidic cell culture device of claim 1, wherein the number of inflow channels is at least 1.
3. The microfluidic cell culture device of claim 1, wherein the number of outflow channels is at least 3.
4. The microfluidic cell culture device of claim 1, wherein the number of aggregate outflow channels is 1.
5. The microfluidic cell culture device of claim 1, wherein the inflow channel is rectangular in cross-section.
6. The microfluidic cell culture device for simulating a filter cartridge of claim 5, wherein the length of the long side of the rectangle is 7-10 mm and the length of the short side is 1-3 mm.
7. The microfluidic cell culture device of claim 1, wherein the outflow channel is circular in cross-section.
8. The microfluidic cell culture device simulating a filter cartridge of claim 7, wherein the diameter of the circle is 180-230 μm.
9. The microfluidic cell culture device simulating a filter cartridge of claim 1, wherein the top of the cell culture chamber is provided with a glue injection hole.
10. The microfluidic cell culture device of claim 1, wherein the cell culture body is further provided with a liquid injection hole and a liquid discharge hole.
11. The microfluidic cell culture device of claim 10, wherein the infusion port is connected to an infusion tube, and the infusion tube is connected to a constant-speed injector.
12. The microfluidic cell culture device of claim 10, wherein the drain hole is connected to a drain tube.
13. The microfluidic cell culture device of claim 1, wherein the cell culture body comprises a silica gel.
14. The microfluidic cell culture device of claim 1, wherein the cover plate comprises a preparation material comprising PMMA.
15. A method of using the microfluidic cell culture device for simulating a filter cartridge of any one of claims 1 to 14, the method comprising:
mixing the cells with the hydrogel, and injecting the mixture into a cell culture cavity through a glue injection hole;
covering a cover plate and a fixing device;
regulating the flow rate of the constant-speed injector, injecting the perfusion liquid from the perfusion tube, collecting and discharging the waste liquid in the liquid discharge tube, and simulating the fluctuation of intraocular pressure by regulating the perfusion pressure;
culturing the cells and observing;
the hydrogel comprises 5% -15% of gelatin by mass, and the change of extracellular matrix and tissue hardness in the process of scar formation of the filtration bleb is simulated by adjusting the hardness of the hydrogel.
16. The method of claim 15, wherein the step of using the microfluidic cell culture device is performed by using a filter membrane,
the density of the cells in the hydrogel is (1-5) multiplied by 10 2 mu.L.
17. The method of claim 15, wherein the constant-speed injector has a flow rate of 0.5-2 mL/day.
18. Use of a microfluidic cell culture device of a simulated filtration pore according to any one of claims 1 to 14 and/or a microfluidic cell culture device of a simulated filtration pore according to any one of claims 15 to 17 in the preparation of a cell dynamic model under simulated filtration pore conditions.
19. The cell dynamic model under the condition of the simulated filtration membrane is characterized in that the cell dynamic model under the condition of the simulated filtration membrane is obtained by culturing the simulated filtration membrane microfluidic cell culture device according to any one of claims 1-14 and/or the simulated filtration membrane microfluidic cell culture device according to any one of claims 15-17.
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