KR20170071450A - 3D cell culture scaffold and co-culture method using the same - Google Patents

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

[0001] The present invention relates to a three-dimensional cell culture scaffold and a co-culture method using the same,

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 used extensively to screen potential drugs rapidly and efficiently. Three-dimensional cell culture methods and organ-on-a-chip devices have been developed to better simulate in vivo 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. 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, 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 .; Lymphocytes (LF) and epithelial cells (ECs) were co-cultured in vitro on a chip in 2013, 13 (8), 1489-500, And can be generated. 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 hydrogel containing cells is put in a membrane form by using surface tension in an O-shaped loop, and it is used as a cell culture dish (plate) The present invention also provides a method for co-culturing a fluid device by dipping the same into a fluid device, 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 days 1 and 10 of culturing cells contained in a hydrogel film formed inside a loop type scaffold.
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 adjusted because the hydrogel-cell mixture is dependent on the diameter size of the loop, and when the diameter of the inner monolithic 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 cells, 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.

Also, when the diameter of the inner single closed curve of the scaffold of the present invention is 3.5 mm to 4.0 mm, 10 to 30 mu l of the hydrogel inside can be collected, and less than 10 mu l can form the hydrogel film according to the surface tension , And more than 30 [mu] l flows out of the membrane and is not preferable for hydrogel film formation.

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. Cell attachment oligopeptide matrix, disclosed in Biomaterials , 2009, in press , 4 V. Jayawarna, et al .. Acta Biomaterialia , 2009, in press , and is not limited as long as it is a gel permeable to air and medium while attaching or supporting 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 claim 1, wherein the scaffold for secretory factor secretion promoting angiogenesis in wells or reservoirs connected to the micro-channel and the medium of the microfluidic device is immersed The present invention relates to a method for manufacturing a blood vessel module.

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 1,100 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) with 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. 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 [mu] L 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 hard, 200 [mu] l of the medium was filled into 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 day 1 and day 10 of culture. Specifically, LF survival in LF-hydrogel loops when incubated with live / dead assay kits (Molecular Probes® L3224, USA) was quantitated. The LF-hydrogel loops were rinsed gently with PBS and immersed in the antibody for 30 minutes. Hoechst 33342 was used to stain nuclei. The LF-hydrogel loops were then rinsed with PBS. Fluorescent images were taken at 3 μm intervals using a confocal microscopy FV1000 (Olympus, Japan). The photographed images were deconvoluted using IMARIS (Bitplane, Switzerland) to create a three-dimensional image.

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 ( μBVM 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 min 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, capillaries formed in microchannels 2 mm to 4.5 mm in length were confirmed within 5 days of culturing. By confirming the existence of microbeads inside the capillaries through the deconvolution image of μBVM, the perfusion ability of μBVM (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 (20)

A three-dimensional cell co-culture apparatus comprising a scaffold for cell culture, and a cell culture plate or a microfluidic device,
(i) the scaffold for cell culture comprises: (a) a scaffold in the form of a single closed curve; And (b) a porous hydrogel formed in film form along the interior monolithic surface of the scaffold, the porous hydrogel comprising a first cell, which permits migration of medium and air but does not allow cell migration;
(ii) said cell culture plate or microfluidic device comprises a second cell; And
(iii) immersing the cell culture dish scaffold in the cell culture plate or the microfluidic device, and co-culturing the first cell and the second cell.
3 - D cell cell culture device. The apparatus of claim 1, wherein the scaffold further comprises a medium for the first cell culture. The three-dimensional cell co-culture apparatus according to claim 1 or 2, wherein the first cell and the second cell are physically separated and cultured. 3. The composition of claim 1 or 2, wherein the hydrogel is selected from the group consisting of fibrin, fibrinogen, collagen, or a polyester polymer, polylactic acid, polyglycolic acid, polycaprolactone, copolymers of polylactic acid- glycolic acid, polyhydroxybutyric acid, Wherein the biodegradable synthetic bioel is a biodegradable synthetic biogel comprising at least one copolymer selected from the group consisting of polyhydroxybutyric acid, polyhydroxy valeric acid, and polyhydroxybutyric acid-valeric acid. The three-dimensional cell co-culture apparatus according to any one of claims 1 to 5, wherein the first cells include 500 cells / 내지 to 6,000 cells / 기준 based on the total volume of the hydrogel. The three-dimensional cell co-culture apparatus according to claim 1 or 2, wherein the first cell is a secretory factor-secreting cell that donates a paracrine factor to the second cell. The method of claim 1 or 2, further comprising dipping the scaffold for cell culture in the cell culture plate or the microfluidic device while co-culturing the first cell and the second cell, Lt; / RTI > cells. 8. The method according to claim 7, wherein the type of the first cell is changed while the cell culture plate or the microfluidic device is immersed and removed in the cell culture plate or the microfluidic device, . A method for producing a scaffold for cell culture according to claim 1,
(i) mixing the first cell and the hydrogel;
(ii) forming the first cell and the hydrogel mixture in a membrane form using surface tension on an inner single closed curve of a single closed curve shaped scaffold;
(iii) solidifying the hydrogel film formed inside the scaffold and containing the first cells; And
(iv) incubating the solidified hydrogel of (iii) with a culture medium. A scaffold for cell culture, prepared according to the method of claim 9. A three-dimensional cell co-culture method comprising immersing a cell culture scaffold containing a first cell in a cell culture plate or a microfluidic device containing a second cell,
The cell culture scaffold comprises (a) a scaffold in the form of a single closed curve; And (b) a porous hydrogel formed in a membrane form along an interior monolithic surface of the scaffold, wherein the porous hydrogel comprises the first cell and permits migration of medium and air but does not allow cell migration.
Three - dimensional cell culture method. 12. The method of claim 11, wherein the scaffold further comprises a medium for the first cell culture. 13. The method according to claim 11 or 12, wherein the first cell and the second cell are physically separated and cultured. The hydrogel according to claim 11 or 12, wherein the hydrogel is selected from the group consisting of fibrin, fibrinogen, collagen, or a polyester polymer, polylactic acid, polyglycolic acid, polycaprolactone, copolymers of polylactic acid-glycolic acid, polyhydroxybutyric acid, Wherein the biodegradable synthetic bioel is a biodegradable synthetic biogel comprising at least one copolymer selected from the group consisting of polyhydroxybutyric acid, polyhydroxy valeric acid and polyhydroxybutyric acid-valeric acid. The method according to claim 11 or 12, wherein the first cells include 500 cells / 내지 to 6,000 cells / 기준 based on the total volume of the hydrogel. 13. The method according to claim 11 or 12, wherein the first cell is a secretory factor-releasing cell that donates a paracrine factor to the second cell. 13. The method according to claim 11 or 12, further comprising immersing the scaffold for cell culture in the cell culture plate or the microfluidic device and then removing the scaffold for the cell culture. 18. The method according to claim 17, wherein the type of the first cell is changed while the cell culture plate or the microfluidic device is immersed and removed in the cell culture plate or the microfluidic device, . 17. The method according to claim 16, wherein the secretion factor donated by the first cell is transferred to the second cell through a medium containing the cell culture scaffold. (i) injecting a hydrogel containing vascular endothelial cells into the micro-channel of the microfluidic device to solidify; And
(ii) immersing the scaffold for cell culture according to claim 8 comprising a micro-channel of the microfluidic device and a secretory factor-secreting cell that promotes angiogenesis in wells or reservoirs connected to the medium.
A method of manufacturing a vascular module.

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