KR20160111683A - 3d cell culture scaffold and co-culture method using the same - Google Patents
3d cell culture scaffold and co-culture method using the same Download PDFInfo
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Abstract
The present invention relates to a scaffold for three-dimensional cell culture in which a hydrogel containing cells is formed in a membrane form by using surface tension in an O-shaped loop, a method for co-culturing cells using the same, and a method for manufacturing a blood vessel module .
Description
The present invention relates to a scaffold for three-dimensional cell culture, a co-culture method using the scaffold, and a method for manufacturing a vascular module.
In vitro cell culture methods have been extensively used to screen potential drugs rapidly and efficiently. 3D cell culture method and a long-term chip (organ-on-a-chip ) the device is in vivo (in RTI ID = 0.0 > vivo < / RTI > conditions. Microfluidics have been used to provide a novel approach to drug screening in order to overcome the limitations of conventional two-dimensional bit analysis (Chung, BG; Lee, KH; Khademhosseini, A .; et al. Lab on a chip 2012, 12 (1), 45-59. 2. Huang, CP; Lu, J .; Seon, H .; et al., Lab on a chip 2009, 9 (12), 1740-8.). By physically and chemically precisely controlling the microenvironment of the cells, microfluidic devices have evolved into sophisticated single organ-chip systems such as the intestine, lung, kidney, and other organs (Huh, D .; Matthews, BD; Mammoto, A .; et al., Science 2010, 328 (5986), 1662-1668. 4. Jang, KJ; Suh, KY, Lab on a chip 2010, 10 (1), 36-42. 5. Kim, HJ; Huh, D .; Hamilton, G .; et al., Lab on a chip 2012, 12 (12), 2165-74.). In addition, a body-on-a-chip platform has been proposed as a toxicity analysis method.
Blood vessels, which are the most important conduits in the organs, have recently been designed on microfluidic platforms (Barkefors, I .; Thorslund, S.; Nikolajeff, F .; et al., Lab on a chip 2009 , 9 , 529-535.). Kim, S .; Lee, H .; Chung, M .; et al., Lab on a chip 2013, 13 (8), 1489-500 have reported that co-culture of lung fibroblasts (LF) and epithelial cells (ECs) can produce a perfusable vascular network in vitro have. In the case of a microfluidic device model for in vivo vessel formation, cells that excrete growth factors inducing angiogenesis, such as lung fibroblasts, dermal fibroblasts, and cancer cells, are co-cultured in a region defined to assist in the assembly of vascular endothelial cells .
In the existing multicellular co-culture, bito assay, two cells coexist from the beginning to the end of the experiment. In addition, there may be physical interference between cells, which also reduces the reliability of the results. Therefore, it is possible to substitute a conditioned medium containing growth factors for a similar effect without co-culturing. However, since the absence of actual culture-supporting cells deteriorates the function of the cell of interest, I did not.
In the case of a bit-lobe model based on a microfluidic device such as a conventional organ-on-a-chip, since the cells exhibiting the paracrine effect are permanently arranged and co-cultured, Irreversible design that can not be altered or removed from the arrangement and configuration of the cells until the end point. However, culture-assisted cells that secrete a paracrine factor outside of the cell of interest at the time of analysis may be a factor that may mislead the response to drugs and the like. Therefore, it is necessary to change the cell microenvironment necessary for development to a form suitable for tissue stabilization, and therefore, a deformable co-cultivation technique is required.
Accordingly, the present invention provides a scaffold for three-dimensional cell culture in which a hydrogel containing cells is put in a membrane form using surface tension in an O-shaped loop, and the scaffold for cell culture is provided with a cell culture dish or a microfluidic device The present invention also provides a method for co-culture by dipping and a method for manufacturing a blood vessel module using the same.
Since the scaffold for three-dimensional cell culture of the present invention contains cells three-dimensionally in a hydrogel, it is cultured in an environment similar to that of a living body. Therefore, the scaffold exhibits high survival rate for a long period of time and is provided with a medium have. When the cell culture technique is used, the hydrogel isolated in the loop acts as a physical barrier to the cells, and has a great advantage that the microenvironment such as the growth factor provided by the cell can be formed without interference. The co-culturing method using the scaffold for three-dimensional cell culture of the present invention can be implemented for all cells capable of culturing on the hydrogel structure. In the dipping in and out method, Which can create a dynamic micro environment in the body, enabling a system design similar to a living body or a disease. Therefore, it can be used as a method for replacing animal experiment and clinical experiment by applying to long - term chip in the future.
FIG. 1 is a schematic diagram showing a process of forming a hydrogel containing LF (lung fibroblasts) cells in a membrane form using surface tension inside a loop-shaped scaffold.
FIG. 2 is a view showing live-dead cell survival rates on
FIG. 3 is a schematic diagram illustrating a process of forming a micro-scale blood vessel module (μBVM) in a micro-channel of a microfluidic device.
FIG. 4 is a diagram showing a bright field image of HUVEC (human umbilical vein endothelial cells) forming a capillary vessel inside a micro-channel by co-culture date with LF.
FIG. 5 is a view (micrometer) showing the inflow of the micro-beads into the blood vessels by photographing the immune-stained μBVM and the micro-beads with a confocal microscope and then deconvoluting them into three-dimensional images.
FIG. 6 is a schematic showing the expansion of a μBVM formed using the present invention and culturing of tissue models of various organs connected to blood vessels with hydrogel loops containing different cells.
Hereinafter, the present invention will be described in detail. However, it should be understood that the present invention may be embodied in many other specific forms without departing from the spirit or essential characteristics thereof.
In one aspect, the present invention provides a scaffold in the form of a single closed curve; And a hydrogel in the form of a membrane formed along the surface of the inner single closed curve of the scaffold.
In one embodiment, the scaffold may be looped.
In one embodiment, the membrane can be three-dimensionally embedded in the hydrogel.
In one embodiment, the number of cells can be controlled because the hydrogel-cell mixture is dependent on the diameter size of the loop, and when the diameter of the inner single closed curve of the scaffold is 3.5 mm to 4.0 mm, The number of cells may be 2 x 10 4 or more and less than 6 x 10 4 cells. When the diameter of the inner single closed curve of the scaffold of the present invention is 3.5 mm to 4.0 mm, when the number of cells contained in the internal hydrogel film is less than 2 × 10 4 , secretion of the secretion factor is undesirably low, When the hydrogel is solidified at 6 × 10 4 or more, the gel is not shrunk and fixed to the loop when the gel is solidified.
When the diameter of a single closed curve of the scaffold of the present invention is 3.5 mm to 4.0 mm, 10 to 30 μl of the internal hydrogel can be collected, and less than 10 μl of the hydrogel can form a hydrogel film And more than 30 flows out of the membrane, which is not preferable for forming a hydrogel film.
The size and volume of the loops of the present invention were optimized for HUVEC angiogenesis experiments using LF as a secretory factor (vascular endothelial growth factor) secretory cell, and other arrangements can be chosen for specific applications. The main advantage of locating the LF secreted by the secretory factor in the loop is that the cells are easily removable, unlike the LF, which was conventionally permanently located throughout the experiment in the microfluidic device. As a result, both types of cells are co-cultured while eliminating physical interferences and contamination of each other, and the effect of two types of cells can be blocked at a desired time. In one embodiment of the present invention, the LF is trapped in the hydrogel in the loop, thereby promoting the growth of HUVEC present in the micro-channel of the microfluidic device to form blood vessels and removing the loop, The influence of secretory factors can be immediately released.
In the present invention, the term "hydrogel " refers to a substance in which a liquid containing water as a dispersion medium through a sol-gel phase transition is hardened to lose fluidity and to have a porous structure, and in the case of a hydrogel suitable for cell attachment and cultivation, It is not limited.
In one embodiment, the hydrogel may be permeable to the medium and air.
In one embodiment, the hydrogel may be fibrin, fibrinogen or collagen, and a biodegradable synthetic biogel may be used. The biodegradable synthetic biogel may be selected from the group consisting of polyester polymers, polylactic acid, polyglycolic acid, polycaprolactone, copolymers of polylactic acid-glycolic acid, polyhydroxybutyric acid, polyhydroxyvaleric acid and polyhydroxybutyric acid-valeric acid And may include one or more kinds of copolymers.
In one embodiment, in addition to the biodegradable synthetic bioel, a porous gel to which cells can attach can be used. For example, the HydroMatrix ™ Peptide Cell Culture Scaffold, DA Narmoneva, et al., Manufactured by Sigma-Aldrich, / Biomaterials 26 (2005) 4837-4846, hydrogel disclosed in WO2007 / 029003 Hydrogel disclosed in US20070099840, M. Zhou et al., Supra. Biomaterials , 2009, in disclosed in press cell attachment matrix oligopeptide, 4 Jayawarna V., et al .. Acta Biomaterialia , 2009, in press , and the like, and it is not limited as long as it is a gel permeable to air and a medium while adhering or supporting the cells.
In one aspect, the present invention provides a method of preparing a cell comprising: 1) mixing cells and a hydrogel; 2) forming a membrane and a mixture of cells and hydrogels in a single closed curve of the scaffold in the form of a single closed curve using surface tension; 3) a method for producing a scaffold for three-dimensional cell culture comprising solidifying a cell-containing hydrogel film formed inside a scaffold.
In one aspect, the present invention relates to a method for producing a three-dimensional cell culture according to the first aspect, wherein the scaffold for three-dimensional cell culture comprising the first cell in a hydrogel is immersed in a cell plate or microfluidic device culture medium in which the second cell is cultured, And a method for co-culturing the cell and the second cell.
In the present invention, the term "paracrine factor" refers to a substance that is secreted in the own cell and acts as an exogenous signal to the surrounding cells to affect the growth of itself and other peripheral cells, for example, an angiogenic factor angiogenesis factor, vascularendothelial cell growth factor, neurotransmitter, cell proliferation factor, cytokinin, growth factor, differentiation factor, autacoid, and the like.
In one embodiment, the first cell may be a donor cell donating a secretagogue factor, and the second cell may be a cell that receives a secretagogue factor from the first cell. In one embodiment of the present invention, a secretory factor (for example, vascular endothelial cell growth factor) secreted by the first cell, LF, promotes the growth of the second cell, HUVEC, to form blood vessels.
In one embodiment, a secretagogue factor secreted in the first cell can be delivered to the second cell via a medium containing a scaffold for three-dimensional cell culture.
By collecting the cells in the form of a hydrogel membrane inside the loop, it is possible to transfer the secretory factors secreted by the first cell to the second cell during cell co-culture, and physically separate it to prevent intercellular contamination due to co- Is easily immersed in a reservoir of a microfluidic device and can be easily taken out, thereby separating two types of cells at any time.
In one aspect, the present invention provides a method of preparing a microfluidic device, comprising: 1) injecting a hydrogel containing vascular endothelial cells into a micro-channel of a microfluidic device to solidify the microgel; And 2) immersing the scaffold for three-dimensional cell culture according to
In one embodiment, the blood vessel module may be a microscale.
In one embodiment, the blood vessels of the vascular module formed in the method may be perfusable.
The present invention will be described in more detail with reference to the following examples. However, the following examples are only for the purpose of illustrating the present invention, and thus the present invention is not limited thereto.
[ Example ]
Example 1. Micro-channel fabrication
Xia, Y .; Whitesides, G. M., SOFT LITHOGRAPHY. Annual Review of Materials Science 1998, 28, 153-184. A microfluidic device was fabricated by soft lithography. Specifically, the plasma-treated silicon wafer was spin-coated with a negative photoresist SU-8100 (Microchem, USA) having a thickness of 150 mu m. After pre-baking at 65 ° C for 10 minutes, 95 ° C for 30 minutes, the wafer was exposed to 405 nm UV (Shinu MST, Korea) for 500 mJ. After exposure, the wafer was baked at 65 占 폚 for 1 minute and at 95 占 폚 for 10 minutes. An unexposed portion of the photoresist was removed using a SU-8 developer (Microchem, USA). The PDMS precursor, Sylgard 184 (Dow Corning, USA) was poured, baked and replicated using the wafer as a master mold. After the inlets and reservoirs were punched out, the PDMS block was attached to the cover slip by treating with plasma (Femto Science, Korea). The attached device was heated in a drying oven at 80 ° C overnight to become hydrophobic on the surface.
Example 2. Cell culture
Human umbilical vein endothelial cells (HUVEC) (Lonza, Switzerland) were cultured in EGM-2 (endothelial growth medium, Lonza, Switzerland) medium, and passage 4 to 5 cells were used . Primary human lung fibroblasts (LF) (Lonza, Switzerland) were cultured in FGM-2 (Fibroblast growth medium, Lonza, Switzerland) medium, and cells with passage number of 6 to 10 were used for the experiments. To obtain the cells, rinsed with PBS and treated with 0.25% trypsin-EDTA (Gibco, USA). Two minutes later, the enzyme action was neutralized by adding M199 (Lonza, Switzerland) containing 10% fetal calf serum. Cells were collected, centrifuged at 1100 rpm for 2 min, and EGM-2 was added to dilute and float to reach the desired number of cells.
Example 3. LF - Hydrogel Loop manufacturing
As shown in FIG. 1, a hydrogel containing LF (lung fibroblasts) was formed into a film shape on the inner surface of a loop-shaped scaffold using surface tension. Specifically, a sterile looped plastic rod (Loop and Needle, SPL, Korea) having an outer diameter of 6.5 mm and an inner diameter of about 3.8 mm was trimmed and used to collect the LF and fibrin mixture. LF was cultured in a tissue culture dish as cells secreting a paracrine factor and was suspended in EGM-2 medium to a concentration of 4 x 10 cells / ml. The bovine fibrinogen solution (2.5 mg / ml fibrinogen containing 0.15 U / ml aprotinin) was added to the cell suspension and mixed with thrombin (0.5 U / ml) just prior to loading into the loop portion. Fibrin (hydrogel with LF) containing a total of 20 μl of LF was gently wrapped around the inner surface of the plastic loop. The gel loaded loops were placed in 96 wells and allowed to gel for 5 minutes. When the gel was hardened, 200 쨉 l of the medium was filled in the wells and incubated for one day. In this example, 20 μl of a hydrogel mixture containing 3 × 10 6 cells / ml of cells was collected using a loop of 3.8 mm in inner diameter. Since the hydrogel-cell mixture is dependent on the diameter size of the loop, Lt; / RTI > However, when hydrogels containing 30,000 or 60,000 LF cells were collected in a loop, when the hydrogel containing 60,000 cells was collected, the gel contracted more than 30,000 cells and the gel was released in the loop .
Example 4. Live / dead analysis
Long-term survival of LF cultured in the loop is essential so that LF continuously secretes growth factors. Therefore, live / dead analysis was performed using loop samples that captured LF - containing fibrin, confirming and comparing cell survival on
As a result, it was found that the cells in the gel collected in all the loops survived, and 95% of the cells survived 10 days after the incubation (DIV 10), showing little difference from DIV 1 (FIG. 2) .
Example 5. Micro Scale Vascular Module (μ BVM Production
In order to confirm the ability of the LF-hydrogel loop to secrete viable blood vessels by promoting HUVEC growth of microfluidic devices co-cultured with LF, human umbilical vein endothelial cells were incubated with fibrinogen Mixed and injected into the micro-channel. The surface tension at the corners of the hydrophobic microchannel filled the HUVEC-containing gel only into the micro-channel area without further progress. The hydrogel was incubated at room temperature for 5 minutes to solidify, and then the medium was added. In Example 3 The prepared LF-hydrogel loops were placed in each reservoir as shown in FIG. 3 and replaced every two days with a fresh medium HUVEC angiogenesis was induced for 5 days and bright field images were obtained.
As a result, it was confirmed that HUVEC formed capillary blood vessels inside the micro-channels by co-culturing with LF cells contained in the hydrogel captured inside the loop (FIG. 4).
Example 6. Immunostaining and Imaging Identification of perfusion characteristics of capillaries through
The capillaries formed in Example 5 were mixed with 4% paraformaldehyde. This was then permeabilized with 0.15% Triton X-100 for 15 minutes and treated with PBS solution containing 3% BSA (Sigma). FITC-conjugated Phalloidin (Molecular Probes, USA) was used to visualize the vessel lumen. Nuclei were stained with Hoechst 33342 (Molecular Probes, USA). Fluorescent images of the capillaries were photographed with an Olympus FV-1000 confocal microscope, and images photographed using IMARIS were deconvoluted into three-dimensional images. In addition, the perfusability of micro-scale blood vessel module (μBVM) was confirmed by introducing 7 μm microbeads showing red fluorescence into the reservoir.
As a result, the capillaries formed in microchannels of 2 mm to 4.5 mm in length were confirmed within 5 days of culturing, and microvoids were observed in the capillaries through the deconvolution image of μBVM, perfusability) (Fig. 5).
In order to confirm whether capillary formation as described above is due to a secretory factor secreted by LF, the LF-hydrogel loop was separated from the reservoir, resulting in degradation of the lumen in one day because no vascular maturation-related growth factor was supplied (Data not shown).
It can be seen from the above results that the co-culture can be performed in a state where the cells in the cell-hydrogel loop and the cells in the microfluidic device are physically separated. In addition, A body-on-a-chip platform can be constructed using a membrane-captured loop of a hydrogel containing cells capable of forming a specific tissue. By changing the type of cells captured in the hydrogel loops of the present invention, the secretion factors exposed to blood vessels can also be changed. Thus, it can be used to study the interaction of blood vessels with other specific cells or tissues, and specific cell-hydrogel loops can be used to co-culture other types of cells that require a side effect factor effect in a physically separate state And can be useful for tissue and organ models in microfluidic research. 6, when the specific cell-hydrogel loop of the present invention and a microfluidic device in which a blood vessel capable of perfusion is formed by using the specific cell-hydrogel loop of the present invention, the μBVM including the biologically- It can be used as a body-on-a-chip platform because it can connect an organ model (a number of cultivated dishes connected in series).
Claims (12)
And a hydrogel in the form of a film formed along a surface of a single closed curve of the scaffold.
2) forming a membrane and a mixture of cells and hydrogels in a single closed curve of the scaffold in the form of a single closed curve using surface tension;
And 3) solidifying the cell membrane-containing hydrogel film formed inside the scaffold.
2) immersing the scaffold for three-dimensional cell culture according to claim 1, comprising a secretory factor-secreting cell that promotes angiogenesis in wells or reservoirs connected to the micro-channel and medium of the microfluidic device Of the vascular module.
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| CN109694825A (en) * | 2019-02-20 | 2019-04-30 | 湖南省肿瘤医院 | A kind of mold for cultivating 3D cell ring |
| KR20190102345A (en) | 2018-02-26 | 2019-09-04 | 강원대학교산학협력단 | Hydrogel membrane fixed vertically in microfluidic chip and preparation method thereof |
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| KR20190102345A (en) | 2018-02-26 | 2019-09-04 | 강원대학교산학협력단 | Hydrogel membrane fixed vertically in microfluidic chip and preparation method thereof |
| CN109694825A (en) * | 2019-02-20 | 2019-04-30 | 湖南省肿瘤医院 | A kind of mold for cultivating 3D cell ring |
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