CN206289263U - Hepatocyte heterogeneity culture apparatus - Google Patents
Hepatocyte heterogeneity culture apparatus Download PDFInfo
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- CN206289263U CN206289263U CN201621287323.4U CN201621287323U CN206289263U CN 206289263 U CN206289263 U CN 206289263U CN 201621287323 U CN201621287323 U CN 201621287323U CN 206289263 U CN206289263 U CN 206289263U
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Abstract
The utility model provides a kind of hepatocyte heterogeneity culture apparatus, including induces chip and the micro-injection pump being connected with the induction chip；The induction chip includes the cell induction region for forming the divided fluid stream passage of concentration gradient and being communicated with the divided fluid stream passage；The heterogeneous distribution of external evoked hepatocyte can be realized by this programme.
Description
Technical field
The utility model is related to field of biomedicine technology, and specifically, the utility model is related to a kind of hepatocyte
Heterogeneous culture apparatus.
Background technology
In terms of research liver cell physiological function in vitro, lot of domestic and foreign scholar employs various methods, such as three
Mingzhi's culture, spherical aggregation culture, micro-fluidic chip culture and 3D printing culture etc., which kind of method its purpose is all to the greatest extent
May substantially hepatic cell growth bad border in analogue body so that when the liver cell of in vitro culture has closer to tumor growth
Physiological function.Have confirmed various dimensional cultures and co-culture research and be substantially better than ordinary flat culture and single culture, but
Heterogeneous this basic physiological feature of internal liver cell is ignored in above-mentioned numerous researchs substantially.Liver minimal structure unit is that liver is small
Leaf, each lobuli hepatis is made up of numerous streak liver plates, and the liver cell in each liver plate near branch of portal vein region is referred to as 1
Area liver cell, 3 area liver cells are referred to as near the liver cell of central vein, between the two referred to as 2 area liver cells.Different zones liver is thin
Born of the same parents physiological function, metabolic capability, have different to aspects such as the sensitiveness of foreign substance the characteristics of, it is different that this is referred to as liver cell
Matter.Wherein, 1 area liver cell in terms of decomposition of glycogen, gluconeogenesis, urea synthesizing, cholesterol biosynthesis, II phase enzyme material metabolism more
Increase effect, 3 area liver cells are in terms of glucose uptake, glycogen storage, glutamine synthesis, alcohol degradation, I phase enzyme material metabolism
It is more efficient.
So being based on bionics thinking, the liver cell that the heterogeneous distribution of liver cell in analogue body is built in vitro is trained in vitro
The method of supporting is significant.
Utility model content
Primary and foremost purpose of the present utility model aims to provide a kind of hepatocyte heterogeneity culture apparatus；
Secondary objective of the present utility model aims to provide a kind of hepatocyte heterogeneity cultural method.
To achieve these goals, the utility model provides following technical scheme：
In a first aspect, the utility model provides a kind of hepatocyte heterogeneity culture apparatus, including induction chip and with
The micro-injection pump that the induction chip is connected；The induction chip includes the divided fluid stream passage for forming concentration gradient
And the cell induction region communicated with the divided fluid stream passage.
Wherein, the induction chip is prepared using optical etching technology mold.
Wherein, the induction chip is prepared using dimethyl silicone polymer.
Wherein, the divided fluid stream passage is laid with first, second training being symmetric along chip longitudinally successively
Support base entrance and the split channel for forming concentration gradient culture medium for being connected and communicating with the culture medium entrance.
Further, the split channel at least includes some fraction circulation roads being sequentially communicated, wherein, the first level shunt
Passage provides two and connects to pour into the one of the induction liquid of various concentrations or heterogeneity respectively with first, second culture medium entrance
Level entrance, and form three primary outlets；The entrance of next stage split channel is corresponded with the outlet of upper level split channel
Connection, and the next stage split channel export volume than the upper level split channel export volume more than one.
Further, the split channel also includes second level split channel and third level split channel；Described second
Fraction circulation road provides three secondary inlets to be connected with three primary outlets respectively, and forms four secondary exit ports；Institute
State third level split channel and provide four three-level entrances to be connected with four secondary exit ports respectively, and form five three-levels
Mouthful.
Wherein, five three-level outlets are arranged side by side and are pooled to the cell induction region.
Wherein, the diameter of the first culture medium entrance and the second culture medium entrance is respectively 500 μm.
Wherein, the first order split channel, second level split channel, the width of third level split channel and depth are equal
It is 100 μm.
Wherein, the cell induction region is in cross, and its vertical passage top is connected with the end of the split channel
And communicate, to be passed through induction liquid；Its vertical passage bottom opens up waste liquid outlet；Its interconnection both sides symmetrically respectively opens up cell
Suspension entrance.
Wherein, the width of the vertical passage is 1mm, and length is 4mm；The width of the interconnection is 1mm, and length is
6.5mm。
Wherein, the vertical passage bottom waste liquid outlet diameter is 750 μm.
Second aspect, the utility model provides a kind of hepatocyte heterogeneity cultural method, using claim 1 to 12
Hepatocyte heterogeneity culture apparatus described in any one.
Wherein, comprise the following steps：
Hepatocyte suspension is added to the cell induction region and the liver cell adherent growth is waited；From the fluid
Split channel continues at the uniform velocity to be passed through the induction liquid of various concentrations or heterogeneity, so as to the Vitro hepatic of the heterogeneous distribution of induced synthesis
Cell.
Further, the hepatocyte origin is in primary hepatocyte.
Wherein, the culture formula of liquid of the liver cell includes DMEM basal mediums, 10% hyclone, 0.5U/ml pancreases
Island element, 7ng/ml hyperglycemic factors, 20ng/ml epidermal growth factors, 7.5 μ g/ml hydrocortisones, 200U/ml penicillin,
200 μ g/ml streptomysins.
Wherein, the cell concentration of the hepatocyte suspension is 1.5 × 107/ml。
Further, before the cell induction region adds the hepatocyte suspension, with 50 μ g/ml under the conditions of 37 DEG C
Fibronectin solution infiltrated.
Preferably, the infiltrating time is 45min.
Wherein, after treating that liver cell is adherent, the entrance of the divided fluid stream passage is closed.
Wherein, it is described induction liquid include WEB nutrient solutions, the WEB nutrient solutions include William ' s E basal mediums,
4mmol/l glutamine, 200U/ml penicillin and 200 μ g/ml streptomysins.
Further, the abductive approach of the heterogeneous liver cell for studying glycometabolism and ammonia metabolism is：From described first
Culture medium entrance pours into the first induction liquid containing hyperglycemic factor 100nmol/l, and is poured into from the second culture medium entrance and contain
The second induction liquid of insulin 100U/L, induction time is 24 hours.
Further, the abductive approach of the heterogeneous liver cell for studying alcohol metabolism and drug metabolism is：From described
First culture medium entrance pours into the WEB nutrient solutions, and is poured into containing 2 μm of 3- methyl courages of ol/l from the second culture medium entrance
3rd induction liquid of anthracene, induction time is 24 hours.
Wherein, after the liver cell of the heterogeneous distribution of induced synthesis, it is additionally included in the heterogeneous culture of the hepatocyte
The research of liver cell physiological function is carried out in device.
Compared to existing technology, scheme of the present utility model has advantages below：
(1) the utility model provides a kind of hepatocyte heterogeneity culture apparatus, and it passes through divided fluid stream passage shape
Cell induction region, the heterogeneous distribution shape of simulation liver cell are passed through after into the induction liquid by micro-injection pump injection of concentration gradient
Into interior environment, the liver cell heterogeneity of its simulation more meets the physiological status of liver cell.
(2) the hepatocyte heterogeneity culture apparatus that the utility model is provided, after the completion of heterogeneous cell induction, may be used also
To proceed physiological Study, using the divided fluid stream passage, the induced drug of input is also formed gradient concentration and be distributed, have
Beneficial to carrying out drug concentration screening.
(3) the utility model also provides a kind of hepatocyte heterogeneity cultural method, and it is heterogeneous that it is specially liver cell
Cultural method, compared to existing other extracorporeal culture of liver cells, the utility model simulates liver cell in vitro under state
The heterogeneous important physilogical characteristics of liver cell, its physiological function that liver cell can be studied under closer to interior state, such as
Glycometabolism, ammonia metabolism, alcohol metabolism and drug metabolism etc., for liver cell physiology, liver tissue engineering research provide new think of
Road.
The additional aspect of the utility model and advantage will be set forth in part in the description, and these are by from following description
In become obvious, or by it is of the present utility model practice recognize.
Brief description of the drawings
The above-mentioned and/or additional aspect of the utility model and advantage from the following description of the accompanying drawings of embodiments will
Become substantially and be readily appreciated that, wherein：
Fig. 1 is the floor layout that a kind of embodiment of the present utility model is used for the heterogeneous culture of hepatocyte.
Fig. 2 is internal liver cell heterogeneity distribution schematic diagram.
Fig. 3 is that the utility model simulation continuous concentration gradient inducing hepatocyte is heterogeneous forms schematic diagram.
Specific embodiment
Embodiment of the present utility model is described below in detail, the example of the embodiment is shown in the drawings, wherein ad initio
Same or similar element or element with same or like function are represented to same or similar label eventually.Below by ginseng
The embodiment for examining Description of Drawings is exemplary, is only used for explaining the utility model, and can not be construed to of the present utility model
Limitation.
Embodiment one
With reference to Fig. 1, the device that the heterogeneous culture of hepatocyte is used for the utility model is described in detail.
In the present embodiment, hepatocyte heterogeneity culture apparatus includes what induction chip 1 was connected with induction chip 1
Micro-injection pump (not shown).
Wherein induction chip 1 includes divided fluid stream passage 11 and the cell for being connected and communicating with the divided fluid stream passage 11
Induced regions 12.
Further, the divided fluid stream passage 11 includes the first culture medium entrance 1101, the second culture medium entrance 1102
And for forming the split channel 111 of concentration gradient nutrient solution.Wherein, two culture medium entrances and split channel 111 are along institute
The induction longitudinally of chip 1 is stated to lay successively.
Further, the first culture medium entrance 1101 and the second culture medium entrance 1102 are symmetric shape, and
It is connected with the micro-injection pump respectively, to pour into induction liquid to the split channel 111.
Further, the split channel 111 at least includes some fraction circulation roads being sequentially communicated, wherein, it is next
The outlet of the entrance of fraction circulation road and upper level split channel is corresponded and connect, and described next collects going out for split channel
Mouthful quantity than the upper level split channel export volume more than one.Specific as follows, first order split channel 1111 provides two
Individual one-level entrance (mark) simultaneously forms three primary outlets (mark), and described two one-level entrances are respectively with first, second
Culture medium entrance 1101,1102 is connected, to pour into the induction liquid of various concentrations or heterogeneity.More preferably, the shunting is logical
Road 111 also includes second level split channel 1112 and third level split channel 1113；The second level split channel 1112 is provided
Three secondary inlets (mark), and form four secondary exit ports (mark), wherein three secondary inlets are respectively with three
The individual primary outlet connection；The third level split channel 1113 provides four three-level entrances (mark), and forms five
Three-level exports (mark), wherein four three-level entrances are connected with four secondary exit ports respectively, five three-levels
Outlet is arranged side by side and is pooled to the cell induction region 12.
In the present embodiment, the decision design three-level split channel of the split channel 111, it is dense that it is mainly formed as needed
Depending on the composition of degree gradient and induction liquid, when under different demands, the design according to the present embodiment for the split channel 111
Scheme can increase or decrease the series of split channel, and the series of split channel is more, and the level of the concentration gradient for being formed is more,
Difference between adjacent concentration value is smaller.The design that series was set up or reduced to the split channel is still model that the present embodiment includes
Enclose, because situation can not excessively be enumerated, the present embodiment explanation preferably by taking three-level as an example.
Preferably, in the present embodiment, the split channel 111 includes that the size of part is as follows：
The diameter of the first culture medium entrance 1101 and the second culture medium entrance 1102 is 500 μm；
First order split channel 1111, second level split channel 1112, the width of third level split channel 1113 and depth
Degree is 100 μm.
Further, the cell induction region 12 is in cross, its vertical passage top and the split channel 111
End connects and communicates, to be passed through the induction liquid (such as Fig. 3) of various concentrations gradient；Its vertical passage bottom opens up waste liquid and goes out
Mouth 122, is mainly used in nutrient solution and the induction liquid freshness for keeping being passed through, and waste liquid is discharged from outlet；Its interconnection two
Side symmetrically respectively opens up cell suspension entrance 121.
In the present embodiment, the size of the part that the cell induction region 12 includes is as follows：
The width of vertical passage is 1mm, and length is 4mm；
The width of interconnection is 1mm, and length is 6.5mm；
Vertical passage bottom waste liquid outlet diameter is 750 μm.
In the present embodiment, hepatocyte heterogeneity culture apparatus is made using photoengraving and molded technology, is adopted
Material is dimethyl silicone polymer, and the device before the heterogeneous culture of liver cell, need to carry out autoclaving, sterilization and bake in vitro
Dry treatment.
Embodiment two
With reference to Fig. 2, the method to the heterogeneous culture of the utility model hepatocyte is described in detail：
Liver minimal structure unit is lobuli hepatis, and each lobuli hepatis is made up of numerous streak liver plates, in each liver plate
Liver cell near branch of portal vein region is referred to as 1 area liver cell 21, and 3 area liver cells are referred to as near the liver cell of central vein
23, referred to as 2 area liver cells 22 between the two.Different zones liver cell is in physiological function, metabolic capability, the sensitivity to foreign substance
Property etc. aspect the characteristics of have different, it is heterogeneous that this is referred to as liver cell.
To carry out hepatocyte heterogeneous for the hepatocyte heterogeneity culture apparatus for illustrating in detail below using embodiment one
The method of property culture.
The equipment that the present embodiment need to be used includes：Cell dissociation equipment, micro-injection pump, syringe, culture dish and glass
Glass rod, above-mentioned equipment is sterilized in super-clean bench using preceding by ultraviolet irradiation.
In the present embodiment, its cell heterogeneity cultural method is specific as follows：
Hepatocyte suspension is added to the cell induction region 12 and the liver cell adherent growth is waited；Wherein, institute
Hepatocyte origin is stated in primary hepatocyte.In the present embodiment, cultivating the culture formula of liquid of the liver cell includes the training of DMEM bases
Support base, 10% hyclone, 0.5U/ml insulin, 7ng/ml hyperglycemic factors, 20ng/ml epidermal growth factors, 7.5 μ g/ml
Hydrocortisone, 200U/ml penicillin, 200 μ g/ml streptomysins.The cell concentration of the hepatocyte suspension is adjusted after its culture
It is 1.5 × 107/ml.Further, before the cell induction region 12 adds the hepatocyte suspension, in 37 DEG C of conditions
The lower fibronectin solution with 50 μ g/ml carries out the infiltration to the cell induction region 12, specifically, during the infiltration
Between be 45min.Further, treat after liver cell adherent growth, add the liver cell to hang in the cell induction region 12
The entrance of liquid is closed, and the entrance is specially two cell suspension entrances 121.
Continue at the uniform velocity to be passed through the induction liquid of various concentrations or heterogeneity from the divided fluid stream passage 11, to induce shape
Into the liver cell of heterogeneous distribution；Further, the induction liquid includes WEB nutrient solutions, and the WEB nutrient solutions include
William ' s E basal mediums, 4mmol/l glutamine, 200U/ml penicillin and 200 μ g/ml streptomysins.
Because the formation of the heterogeneous distribution of liver cell has important to studying its liver cell physiological function under state in vitro
Meaning, and physiological function therein is mainly including glycometabolism, ammonia metabolism, alcohol metabolism, drug metabolism etc..Below to for grinding
Induction and research method required for studying carefully the heterogeneous culture of liver cell of various physiological functions are described in detail：
The abductive approach of the heterogeneous liver cell for studying glycometabolism and ammonia metabolism is：From the first culture medium entrance
1101 pour into the first induction liquid containing hyperglycemic factor 100nmol/l, and are poured into containing pancreas from the second culture medium entrance 1102
The second induction liquid of island element 100U/L, induction time is 24 hours.The first induction liquid, the second induction liquid are by the shunting
The induction liquid (such as Fig. 3) of continuous concentration gradient is formed after passage 111.
Its research method is：Liver cell after induction is carried out into staining for glycogen or aminomethyl phosphonic acid synzyme (CPS1) is exempted from
Epidemic disease fluorescent staining, the heterogeneous influence to glycometabolism and ammonia metabolism of liver cell from left to right in observation chip.
The abductive approach of the heterogeneous liver cell for studying alcohol metabolism and drug metabolism is：From first culture medium
Entrance 1101 pours into the WEB nutrient solutions, and is poured into containing 2 μm of 3-MECAs of ol/l from the second culture medium entrance 1102
The 3rd induction liquid, induction time be 24 hours.The WEB nutrient solutions, the 3rd that the first culture medium entrance 1101 is poured into
The induction liquid (such as Fig. 3) that induction liquid passes through formation continuous concentration gradient after the split channel 111.
Its research method is：
After (1) two culture medium entrance stops being passed through liquid, after sucking residual media, 200 μm of alkene of ol/l are added
The paracetamol that propyl alcohol acts on 2 hours or 10mmol/l is acted on 4 hours.
(2) the liver cell tetramethylrhodamine methyl esters fluorescence signal interior liver cell from left to right of observation chip after detection induction
The heterogeneous influence to alcohol metabolism；The cytochrome pathways subfamily 3A4 in liver cell after detection induction
(CYP3A4) the heterogeneous influence to drug metabolism of liver cell from left to right in observation chip such as.
In sum, master-plan of the present utility model is to prepare poly- diformazan by traditional soft lithographic and again molded technology
Siloxane reactions chip, the hepatocyte suspension after concentration of cell suspension will be adjusted using nutrient solution and is injected in induction chip and house
Wait until within cell culture incubator 3-4 hours that induction liquid is initially added into after cell attachment to be induced.Work of the induction liquid in micro-injection pump
Under, pour into after induction chip because chip is to the diversion design of the logical part of induced flow, form concentration gradient, then persistently fill
The induction liquid for entering is derived automatically from from chip outlet.The timing 24 hours since cell is inoculated with, cell then completes heterogeneous point of cell
The growth of cloth, can be studied thereafter using the no research method physiological function different to cell.
The above is only some embodiments of the present utility model, it is noted that for the common skill of the art
For art personnel, on the premise of the utility model principle is not departed from, some improvements and modifications can also be made, these improve and
Retouching also should be regarded as protection domain of the present utility model.
Claims (12)
1. a kind of hepatocyte heterogeneity culture apparatus, it is characterised in that including induction chip and with the induction chip phase
The micro-injection pump of connection；It is described induction chip include for formed concentration gradient divided fluid stream passage and with the fluid
The cell induction region that split channel is communicated.
2. hepatocyte according to claim 1 heterogeneity culture apparatus, it is characterised in that the induction chip is utilized
It is prepared by optical etching technology mold.
3. hepatocyte according to claim 1 heterogeneity culture apparatus, it is characterised in that the induction chip is used
It is prepared by dimethyl silicone polymer.
4. hepatocyte according to claim 1 heterogeneity culture apparatus, it is characterised in that the divided fluid stream passage
Along the induction chip longitudinally be laid with successively the first, second culture medium entrance that is symmetric and with the culture
The split channel for forming concentration gradient nutrient solution that base entrance is connected and communicated.
5. hepatocyte according to claim 4 heterogeneity culture apparatus, it is characterised in that the split channel is at least
Including some fraction circulation roads being sequentially communicated, wherein, first order split channel provide two respectively with first, second culture medium
Entrance connection forms three primary outlets to pour into the one-level entrance of the induction liquid of various concentrations or heterogeneity；Next stage
The outlet of the entrance of split channel and upper level split channel is corresponded and connected, and the next stage split channel outlet
Quantity than the upper level split channel export volume more than one.
6. hepatocyte according to claim 5 heterogeneity culture apparatus, it is characterised in that the split channel is also wrapped
Include second level split channel and third level split channel；The second level split channel provides three secondary inlets with respectively with three
The individual primary outlet connection, and form four secondary exit ports；The third level split channel provides four three-level entrances to divide
Do not connected with four secondary exit ports, and form five three-level outlets.
7. hepatocyte according to claim 6 heterogeneity culture apparatus, it is characterised in that five three-levels outlets
It is arranged side by side and is pooled to the cell induction region.
8. hepatocyte according to claim 4 heterogeneity culture apparatus, it is characterised in that first culture medium enters
The diameter of mouth and the second culture medium entrance is respectively 500 μm.
9. hepatocyte according to claim 6 heterogeneity culture apparatus, it is characterised in that first level shunt is led to
Road, second level split channel, the width of third level split channel and depth are 100 μm.
10. hepatocyte according to claim 1 heterogeneity culture apparatus, it is characterised in that the cell induction area
Domain is in cross, and its vertical passage top is connected and communicates with the end of the divided fluid stream passage, to be passed through induction liquid；It is indulged
Waste liquid is opened up to passage bottom to export；Its interconnection both sides symmetrically respectively opens up cell suspension entrance.
11. hepatocyte heterogeneity culture apparatuses according to claim 10, it is characterised in that the vertical passage
Width is 1mm, and length is 4mm；The width of the interconnection is 1mm, and length is 6.5mm.
12. hepatocyte heterogeneity culture apparatuses according to claim 10, it is characterised in that the vertical passage bottom
End waste liquid outlet diameter is 750 μm.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106497788A (en) * | 2016-11-28 | 2017-03-15 | 南方医科大学珠江医院 | Hepatocyte heterogeneity culture apparatuses and its cultural method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106497788A (en) * | 2016-11-28 | 2017-03-15 | 南方医科大学珠江医院 | Hepatocyte heterogeneity culture apparatuses and its cultural method |
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