CN108504571B - Device and method for constructing artificial liver lobule functional unit - Google Patents
Device and method for constructing artificial liver lobule functional unit Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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Abstract
The invention discloses a device and a method for constructing a hepatic lobule functional unit. The device comprises a liver plate basal layer, a liver parenchymal cell area and a fluid channel layer, wherein a liver plate patterning groove is processed on the liver plate basal layer, and the fluid channel layer comprises a fluid inlet channel, an endothelial cell directional growth area and a fluid outlet channel. The corresponding construction method comprises the steps of paving collagen containing parenchymal hepatocytes into the patterned groove of the liver plate, bonding the basal layer of the liver plate with the fluid channel layer after the collagen is solidified, filling endothelial cell suspension into the directional growth area of the endothelial cells, and enabling cell culture medium solution to flow in a one-way mode through the fluid inlet channel, the directional growth area of the endothelial cells and the fluid outlet channel after the endothelial cells grow for a certain amount. The invention realizes the real layer-to-layer position relationship between the endothelial layer and the parenchymal hepatic cells under dynamic culture, the parenchymal hepatic cells have more real response to physicochemical and biochemical stimulation under the protection of the vascular endothelial layer, and when the cells are applied to drug screening, case study and therapy detection, more accurate results can be obtained.
Description
Technical Field
The invention relates to the field of liver tissue engineering and bioartificial liver, in particular to a device and a method for constructing an artificial liver lobule functional unit.
Background
Research in tissue engineering and regenerative medicine integrates the application of multiple fields including life sciences and engineering techniques to produce functional human tissue. The development of liver tissue engineering aims to provide living body transplantation for patients with impaired liver function or perform liver function-related function detection, drug metabolism, toxicity test and the like. The traditional construction method is a top-down construction method, namely, liver cells are transplanted to a polymer scaffold and grow directionally under the action of a cell matrix. However, this approach does not allow for a good microstructural construction of an accurate in vitro cell population. In a relative bottom-up construction mode, the building can be carried out from the minimum unit of the liver, and then recombination is carried out to realize pyramid-shaped accumulation.
The liver lobule is a basic unit of liver functionality, has a radial structure in shape, and includes basic metabolic functions, substance transfer, partial immune functions and the like of the liver. At present, Ma Chao et al realize structured co-culture of Liver cells and endothelial cells in a pneumatic model by constructing a set of dual-channel pneumatic microfluidic chips (Chao M, Lei Z, Zhou E M, et al. on-Chip Construction of Liver cell-like microorganism and Its Application for additive Drug Chemistry,2016,88(3): 1719.). Ho et al achieve patterning of hepatic lobular structures by fabricating electrodes on a microchip, selectively adsorbing hepatic and endothelial cells by changes in the site of energization (Ho C T, Lin R Z, Chen R J, et al, liver-cell patterning Lab Chip: microfabrication of liver lobular tissue [ J ]. Lab on A Chip,2013,13(18): 3578-. Ma et al achieve selective area photocuring under mask coverage by mixing cell suspensions with photosensitive materials to make cell colloids (Ma X, Qu X, Zhu W, et al. defined National pattern printed biological man iPSC-derived high-efficiency model via rapid 3Dbioprinting. [ J ]. Proceedings of the National Academy of Sciences of the United States of America,2016,113 (2206)). The existing method for constructing the hepatic lobule functional unit has a serious defect that an endothelial layer and a hepatic parenchymal cell layer are positioned on the same plane, and the actual direction of substance transmission is transmitted to the hepatic parenchymal cell through the endothelial layer, so that the endothelial layer plays a role in protecting the hepatic parenchymal cell to a certain extent, and the hepatic parenchymal cell is prevented from being washed by laminar flow. For the reasons mentioned above, the existing liver lobule model has a certain bias for drug screening and metabolic function detection.
Chinese invention CN105170205B (published: 2017, 1/25) discloses a bionic chip constructed based on microfluidic chip technology, which utilizes porous membrane to stack an elimination module, a distribution module, a drug effect module, a first and a second liver metabolism modules and an absorption module together to form an integral structure from top to bottom for evaluating absorption, distribution, metabolism, elimination, drug effect and liver toxicity of drugs. However, the porous membrane will block the mass transport ability between the two cells to some extent, and will also affect the results of drug screening and metabolic function testing.
Disclosure of Invention
The invention mainly aims to overcome the defects of the prior art and provides a device and a method for constructing an artificial liver lobule functional unit. The hepatic lobule functional unit constructed by the invention simulates the real structure of hepatic lobules, so that the hepatic lobule functional unit has more real experimental data when being used for drug screening and metabolic function detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
a device for constructing an artificial liver lobule functional unit comprises a liver plate basal layer, a liver parenchymal cell area and a fluid channel layer which are sequentially combined together from bottom to top, wherein a liver plate patterning groove is processed on the liver plate basal layer, the fluid channel layer comprises a fluid inlet channel, an endothelial cell directional growth area and a fluid outlet channel, the liver parenchymal cell area is paved in the liver plate patterning groove, cell fillers in the liver parenchymal cell area are in gel shapes, and the tops of the cell fillers are directly exposed at the bottom of the endothelial cell directional growth area.
As an improved technical scheme of the invention, endothelial cell suspension filling channels are arranged in a liver plate basal layer or a fluid channel layer corresponding to an endothelial cell directional growth area, the number of the endothelial cell suspension filling channels is more than or equal to 2, and sealing sheets are also arranged at the openings of the endothelial cell suspension filling channels.
As an improved technical scheme of the invention, a micro-column array is arranged in the liver plate patterning groove, the height of the micro-columns is 10-1000 mu m, the diameter of the micro-columns is 10-500 mu m, the distance between the micro-columns is 10-1000 mu m, and the diameter size of the liver plate patterning groove is 100-10 mm.
As an improved technical scheme of the invention, the directional endothelial cell growth area is divided into a plurality of fan-shaped flow domains by a flow channel wall, and the plurality of flow domains originate from the fluid inlet channel and converge at the fluid outlet channel.
Preferably, the branched drainage basins of the fan-shaped multi-branched drainage basin have the width of 10-1000 μm and the depth of 10-2000 μm. .
Furthermore, the buckling relation between the fluid channel layer and the basal layer of the liver plate is that the geometric centers of the parenchymal hepatic cell area and the directional growth area of endothelial cells correspond to each other.
As another object of the present invention, there is provided a method for constructing an artificial liver lobule functional unit, comprising:
s1, preparing a liver plate basal layer and a fluid channel layer in the artificial liver lobule functional unit constructing device;
s2, paving the collagen mixed with the parenchymal hepatic cells into the patterned grooves of the liver plate, and scraping;
s3, bonding the basal layer of the liver plate with the fluid channel layer after the collagen is gelatinized;
s4, filling the suspension containing the endothelial cells into the fluid channel layer, and sealing the whole device;
and S5, culturing in a culture device, and growing endothelial cells to obtain the artificial liver lobule functional unit.
As an improved technical scheme of the invention, the specific step of S4 is that the fluid inlet channel and the fluid outlet channel are closed, then the suspension containing endothelial cells is filled into the endothelial cell directional growth area through the endothelial cell suspension filling channel, and finally the sealing sheet is used for sealing the opening of the endothelial cell suspension filling channel, thereby closing the whole device.
The improved technical scheme of the invention comprises the following subsequent steps of opening the fluid inlet channel and the fluid outlet channel, introducing the cell culture medium solution into the fluid inlet channel, enabling the cell culture medium solution to flow into the endothelial cell directional growth area from the fluid inlet channel and then flow out from the fluid outlet channel, and enabling endothelial cells and liver parenchymal cells to grow in an environment where the cell culture medium solution flows.
As a further improved technical scheme of the invention, the method also comprises the following subsequent steps of removing the cell culture medium solution, introducing the cell culture medium solution added with the medicine, and enabling endothelial cells and liver parenchymal cells to grow in the flowing environment of the cell culture medium solution added with the medicine to obtain experimental data of medicine screening and metabolic function detection.
As an improved technical scheme of the invention, the flow rate of the cell culture medium solution is controlled to be 0.1-100 mu m/s.
As an improved technical scheme of the invention, the cell filler in the liver parenchymal cell area is prepared by mixing collagen and a liver cell suspension, and the content of the liver cells in a single liver lobule functional unit is 1e2～1e6And (4) respectively.
Further, the collagen has good biocompatibility.
Preferably, the collagen is one or a mixture of a rat tail type I collagen system and a gelatin sodium alginate system, and the final concentration of the collagen is 1-10 mg/ml.
As an improved technical scheme of the invention, the endothelial cell content of the endothelial cell suspension poured into the single hepatic lobule functional unit is 1e2～1e4And (4) respectively.
As an improved technical scheme of the invention, the cell culture medium solution is formed by mixing a liver cell culture medium solution and an endothelial cell culture medium solution, and the volume ratio of the liver cell culture medium solution to the endothelial cell culture medium solution is 0.1-10: 1.
Compared with the prior art, the invention has the following advantages:
(1) the hepatic parenchymal cells and the endothelial cells constructed by the model are in the same layer, the positions of the hepatic parenchymal cells and the endothelial cells constructed by the model are in a spatial structure relationship, and when the density of the endothelial cells reaches a certain degree, the endothelial layer can realize the function of a vascular-like layer.
(2) The previous model does not assemble a lobular model of the liver with power transmission, and the model can realize the promotion effect of fluid on the growth of the endothelial layer and simultaneously protect the parenchymal liver cells from the influence of the fluid shearing force.
(3) In the prior art, the contact culture of two types of cells is realized by adopting structures such as PET, porous silicon and the like, the model directly realizes the model construction of the contact culture of the two types of cells, and the material transmission capability of the model is improved.
In summary, according to the device and the method for constructing the artificial liver lobule functional unit provided by the invention, the constructed liver lobule functional unit comprises co-culture of two essential cells, namely, liver parenchymal cells and endothelial cells, which are most basic in liver lobules, the real layer-to-layer position relationship between the endothelial layer and the liver parenchymal cells is realized under dynamic culture, the response of the liver parenchymal cells to physicochemical and biochemical stimulation under the protection of the blood vessel endothelial layer tends to be more real, and when the liver lobule functional unit is applied to drug screening, case study and therapy detection, a more accurate result can be obtained. In addition, the invention also realizes the construction of a multi-channel laminar flow model, a plurality of independent units are integrated on one chip, a plurality of independent units can be observed simultaneously, and the solution can be extracted intensively for research. For example, when the chip is used for detecting the function of the liver cells, the chip can be damaged, and the liver cells can be independently extracted for further research.
Drawings
FIG. 1 is a schematic view of the bonding structure of the apparatus of the present invention;
FIG. 2 is a schematic view of the split structure of the device of the present invention;
FIG. 3 is a schematic diagram of collagen placement with hepatocytes;
FIG. 4 is a schematic illustration of the perfusion of endothelial cell suspension;
FIG. 5 is a schematic diagram of the distribution of parenchymal hepatocytes and endothelial cells;
FIG. 6 is a microscopic view of distribution of parenchymal hepatocytes and endothelial cells.
Detailed Description
So that those skilled in the art can clearly understand the invention, a detailed description of the invention will be given with reference to the detailed description and the accompanying drawings.
Example 1
Referring to fig. 1-2, the device for constructing an artificial liver leaflet functional unit of the present invention comprises a liver plate basal layer 1, a liver parenchymal cell region 2, and a fluid channel layer 3, which are sequentially combined together from bottom to top, wherein a liver plate patterned groove is processed on the liver plate basal layer 1, the fluid channel layer 3 comprises a fluid inlet channel 31, an endothelial cell directional growth region 32, and a fluid outlet channel 33, the liver parenchymal cell region 2 is laid in the liver plate patterned groove, a cell filler in the liver parenchymal cell region 2 is in a gel shape, and the top of the cell filler is directly exposed at the bottom of the endothelial cell directional growth region 32.
The micro-column array is arranged in the liver plate patterned groove, the height of the micro-columns 11 is 10-1000 mu m, the diameter of the micro-columns 11 is 10-500 mu m, the distance between the micro-columns 11 is 10-1000 mu m, and the diameter of the liver plate patterned groove is 100-10 mm. The endothelial cell directional growth zone is divided by the channel walls 34 into fan-shaped multiple flow domains, which originate from the fluid inlet channel 31 and converge at the fluid outlet channel 33. Preferably, the branched drainage basins of the fan-shaped multi-branched drainage basin have the width of 10-1000 μm and the depth of 10-2000 μm. The buckling relation between the fluid channel layer 3 and the basal lamina of the liver plate 1 is that the geometric centers of the parenchymal hepatic cell region 2 and the directional endothelial cell growth region 32 correspond.
In a preferred embodiment, the liver plate basal layer 1 and the fluid channel layer 3 are both prepared by inverse molding PDMS (polydimethylsiloxane).
Pouring SU-82100 photoresist on the pre-baked silicon wafer, spin-coating at 500rpm and 1500rpm for 10s and 30s, and baking at 65 deg.C and 95 deg.C for 6min and 40 min. Carrying out ultraviolet curing with a mask pattern for 30s on an ultraviolet curing device, and then baking the mask pattern on baking plates at 65 ℃ and 95 ℃ for 6min and 20min respectively. And (3) placing the baked silicon wafer into a developing solution for developing for 5min, cleaning the silicon wafer with alcohol, drying the silicon wafer with nitrogen, and baking the silicon wafer on a baking plate at 150 ℃. Obtaining the groove cake-shaped pattern die containing micropores. In a preferred embodiment, the diameter of the grooved discoid pattern containing the micropores is 2.5mm, the diameter of the micropores is 100 μm, the pitch of the micropores is 300 μm, and the depth of the grooves and the micropores is 400 μm. Pouring 20g of PDMS mixed solution on a groove cake-shaped pattern mould containing micropores, and placing the mould in an oven to bake the mould for 30min at 85 ℃. A liver plate basal lamina 1 was obtained. Holes are punched at the position of the liver basal layer corresponding to the directional endothelial cell growth area 32 to prepare endothelial cell suspension to be filled into the channel 36.
Similarly, SU-82150 photoresist was cast onto pre-baked silicon wafers, spin coated at 500rpm and 1500rpm for 10s and 30s, respectively, and baked at 65 deg.C and 95 deg.C for 10min and 90min, respectively. And (3) performing ultraviolet curing on ultraviolet curing equipment for 30s, then placing the ultraviolet curing equipment on drying plates at 65 ℃ and 95 ℃ for baking for 6min and 20min, repeating the spin-coating and baking steps for 3 times, and respectively performing alignment photoetching by adopting different mask plates. And (3) placing the baked silicon wafer into a developing solution for developing for 5min, cleaning the silicon wafer with alcohol, drying the silicon wafer with nitrogen, and baking the silicon wafer on a baking plate at 150 ℃. Obtaining the fan-shaped multi-branch runner die with the runner depth of 1 mm. In a preferred embodiment, the width of the branch runner corresponding to the fan-shaped multi-branch runner mold is 200 μm, and the diameter of the entire fan-shaped multi-branch runner is 3mm, as shown in fig. 5. Mixing PDMS and a curing agent according to a ratio of 10:1, placing the PDMS mixed solution into a vacuum drying dish after uniform mixing, vacuumizing for 5min, pouring 20g of the PDMS mixed solution onto a template of a fan-shaped multi-branch-basin runner mold, attaching a layer of flat silicon wafer on the PDMS mixed solution to enable the PDMS mixed solution to be positioned in an interlayer, and placing the PDMS mixed solution into an oven to bake for 30min at 85 ℃. The PDMS was then removed to obtain a fluidic channel.
And (3) putting the fluid channel and the glass plate into an oxygen plasma cleaning machine for cleaning for 30min, taking out the fluid channel and the glass plate, bonding the non-working surface of the fluid channel layer 3 with the glass plate, and putting the glass plate into an oven for baking for 30min at 85 ℃ to obtain the fluid channel layer 3.
The liver plate basal layer 1 and the fluid channel layer 3 are washed by alcohol, washed by PBS (phosphate buffer solution), dried in the air and sterilized for 60min under the irradiation of ultraviolet light.
In a preferred embodiment, the plated hepatocyte HepG2 is digested with pancreatin to prepare 1ml of cell suspension, 20 μ L of the cell suspension is taken, 20 μ L of 1X DMEM medium solution is placed in ice to be fully cooled, 3 μ L of 1M NaOH solution is taken and added into an EP tube, 11 μ L of 10X DMEM medium solution is taken and added into the ice to be cooled after being fully mixed, 100 μ L of rat tail type I collagen after being cooled is mixed into NaOH-10X DMEM medium solution and is fully blown to obtain the neutralized rat tail collagen. Ice bath operation the cell suspension and 1 × DMEM medium solution were added to the neutralized rat tail collagen. The collagen concentration was 3.5mg/ml and the cell number per functional unit was 1.0e4。
The prepared collagen 10 μ L is spread into the groove of the liver plate basal lamina 1, and is scraped by a scraper to be spread into the groove, as shown in fig. 3. After being paved, the mixture is placed in an incubator at 37 ℃ for curing for 30 min. And (3) placing the fluid channel layer 3 into an oxygen plasma cleaning machine for treatment for 30s, and bonding the working surface with the liver plate basal layer 1.
Digesting the endothelial cell HUVEC suspension which is paved into 10 mu L of cell suspension by pancreatin, taking 100 mu L of cell suspension, filling the cell suspension into a channel through the endothelial cell HUVEC suspension, adding the cell suspension into the fluid channel layer 3 to ensure that the cell suspension is fully contacted with the hepatocyte HepG2 collagen, cutting the PDMS thin sheet into square thin sheets with the size of 1mm multiplied by 1mm after filling to prepare sealing sheets 35, and blocking the sealing sheets at the inlet of the channel filled with the endothelial cell HUVEC suspension, as shown in figure 4. The chip is placed into an incubator upside down for 12h of incubation.
Mixing the 1X DMEM medium solution and the 1X RPMi medium solution according to a ratio of 1:1, and filling the mixed medium solution into the chip through an external interface so as to enable the mixed medium solution to flow continuously in a single direction. The flow rate was set at 10 μm/s here. After dynamic culture for 2 days in an incubator, the internal medium solution was aspirated out by a pipette gun, and then the 1 × DMEM medium solution and the 1 × RPMi medium solution with the drug added were added to the chip, and the pump was continuously rotated, thereby observing the effect of the drug on the model. The distribution of parenchymal cells and endothelial cells is schematically shown in FIG. 5, and the distribution of parenchymal cells and endothelial cells observed under a microscope is shown in FIG. 6.
Example 2
Example 2 substantially the same as example 1 except that the preparation method of collagen mixed with hepatocyte HepG2 was changed by weighing 1g of gelatin and 0.1g of sodium alginate, pouring into a serum bottle, adding 10ml of pbs solution to the serum bottle with a pipette, stirring the materials with a glass rod to mix them as uniformly as possible, putting the materials into a dry box at 70 ℃, taking out and cooling for 30min every 30min, and repeating 3 times. The concentration of the material was 10% (w/v), and the plated hepatocytes HepG 2100. mu.L were added to the collagen and stirred well. Dropping prepared collagen mixed with hepatocyte HepG2 on the basal layer 1(1) of the liver plate, scraping by using a cell scraper, dropping 1% (w/v) sodium alginate for curing, and carrying out the next operation after the collagen is completely solidified.
It is apparent that the above examples are only examples for clearly illustrating the present invention, and are not to be construed as limiting the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modifications made on the basis of the examples of the present invention, which are common knowledge, are within the scope of the present invention.
Claims (9)
1. The utility model provides a device for constructing artificial liver lobule functional unit, comprises liver board stratum basale, liver parenchymal cell district, fluid channel layer that from bottom to top combines together in proper order, its characterized in that: a liver plate patterning groove is processed on the liver plate basal layer, the fluid channel layer comprises a fluid inlet channel, an endothelial cell directional growth area and a fluid outlet channel, the liver parenchymal cell area is paved in the liver plate patterning groove, cell fillers in the liver parenchymal cell area are in a gel shape, and the top of the cell fillers is directly exposed at the bottom of the endothelial cell directional growth area; a micro-column array is arranged in the liver plate patterning groove, the height of the micro-columns is 10-1000 mu m, the diameter of the micro-columns is 10-500 mu m, the distance between the micro-columns is 10-1000 mu m, and the diameter of the liver plate patterning groove is 100-10 mm; the buckling relation between the fluid channel layer and the basal layer of the liver plate is that the geometric centers of the parenchymal hepatic cell area and the directional growth area of endothelial cells correspond to each other.
2. The apparatus of claim 1, wherein: an endothelial cell suspension filling channel is arranged in a liver plate basal layer or a fluid channel layer corresponding to the endothelial cell directional growth area, the number of the endothelial cell suspension filling channels is more than or equal to 2, and a sealing sheet is also arranged at the opening of the endothelial cell suspension filling channel.
3. The apparatus of claim 1, wherein: the endothelial cell directional growth area is divided into a plurality of fan-shaped flow domains by the flow channel wall, and the plurality of flow domains originate from the fluid inlet channel and converge at the fluid outlet channel.
4. The apparatus of claim 3, wherein: the width of the branch flow field of the fan-shaped multi-branch flow field is 10-1000 mu m, and the depth of the branch flow field is 10-2000 mu m.
5. A method for constructing an artificial liver lobule functional unit is characterized by comprising the following steps:
s1, preparing a liver plate basal layer and a fluid channel layer in the device for constructing the artificial liver lobule functional unit according to any one of claims 1-4;
s2, paving the collagen mixed with the parenchymal hepatic cells into the patterned grooves of the liver plate, and scraping;
s3, bonding the basal layer of the liver plate with the fluid channel layer after the collagen is gelatinized;
s4, filling the suspension containing the endothelial cells into the fluid channel layer, and sealing the whole device;
and S5, culturing in a culture apparatus, and growing endothelial cells to obtain the artificial liver lobule functional unit.
6. The method of claim 5, wherein: s4 comprises the specific steps of firstly closing the fluid inlet channel and the fluid outlet channel, then filling the endothelial cell suspension-containing suspension into the endothelial cell directional growth area through the endothelial cell suspension filling channel, and finally sealing the opening of the endothelial cell suspension filling channel by using the sealing sheet, thereby closing the whole device.
7. The method of claim 5, wherein: comprising the following subsequent steps of opening the fluid inlet channel and the fluid outlet channel, introducing a cell culture medium solution into the fluid inlet channel, flowing the cell culture medium solution from the fluid inlet channel into the endothelial cell directional growth zone and then out of the fluid outlet channel, and growing endothelial cells and hepatocytes in an environment in which the cell culture medium solution flows.
8. The method of claim 7, wherein: the method also comprises the following subsequent steps of removing the cell culture medium solution, introducing the cell culture medium solution added with the medicine, and enabling endothelial cells and liver parenchyma cells to grow in the flowing environment of the cell culture medium solution added with the medicine to obtain experimental data of medicine screening and metabolic function detection.
9. The method of claim 7, wherein: controlling the flow rate of the cell culture medium solution to be 0.1-100 mu m/s.
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| CN110408538B (en) * | 2019-07-18 | 2024-01-26 | 安徽骆华生物科技有限公司 | Liver chip of many liver lobule integrated structure |
| WO2022083037A1 (en) * | 2020-10-19 | 2022-04-28 | 清华大学 | Bionic artificial liver tissue, preparation method therefor and application thereof |
| CN113350574B (en) * | 2021-05-26 | 2022-11-18 | 泸州国之荣耀酒业有限公司 | Method for weaving patterned liver-like small She Weizu |
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