CN107955790A - Intestines-kidney system of aids drug body absorption process based on micro-fluidic chip - Google Patents

Intestines-kidney system of aids drug body absorption process based on micro-fluidic chip Download PDF

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CN107955790A
CN107955790A CN201610896832.5A CN201610896832A CN107955790A CN 107955790 A CN107955790 A CN 107955790A CN 201610896832 A CN201610896832 A CN 201610896832A CN 107955790 A CN107955790 A CN 107955790A
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秦建华
李中玉
陶婷婷
郭雅琼
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Dalian Institute of Chemical Physics of CAS
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Abstract

The present invention provides a kind of intestines kidney system of the aids drug body absorption process based on micro-fluidic chip, and the micro-fluidic chip is mainly made of top layer chip, porous membrane, bottom chip.Top layer chip is inoculated with enterocyte, can be with the absorption process of medicine in analogue body；Bottom chip is mainly connected by left side main channel with right side main channel by collagen passage.Glomerulus micro-assembly robot is inoculated with left side main channel, hemispherical interface of the glomerulus micro-assembly robot in left side main channel and collagen passage is grown by edge-on culture and forms barrier cell, right side main channel is as collecting region, influence of the renal toxicity to detection of glomeruli filtration function caused by the filtration of glomerulus, and chemical composition of the medicine after intestinal absorption can be observed.Different pharmaceutical combination can be observed to toxic action caused by the influence of absorption.The structure of the drug toxicity appraisement system of absorption process in analogue body, and its evaluate application in renal toxicity of the medicine after absorption.

Description

Intestines-kidney system of aids drug body absorption process based on micro-fluidic chip
Technical field
The present invention relates to the technical field that microfluidic chip technology is applied to toxicity assessment system construction, and in particular to base In intestines-kidney system of the aids drug body absorption process of micro-fluidic chip.
Background technology
Zoopery occupies particularly important position in modern medicine and biology, but funds and animal welfare Also into the problem of being difficult to avoid.With reference to microflow control technique and bioscience technology, a kind of " organ chip " has been createed, can The function of human organ is replicated with microchip, medical experiment is become more easy.
It is administered orally as a kind of traditional administering mode, is that people institute is extensive with its opposite convenience and security Receive.Absorption process is one of key factor for determining oral drug bioavailability.And small intestine is the master of oral drug absorption Position is wanted, so mainly studying cross-film of the medicines in small intestine site using various external and in situ intestinal absorptive models both at home and abroad at present Transport process, so as to predict its oral absorption situation.Wherein, Caco-2 cell monolayers model is to be widely used in studying at present One of external model of biological membrane permeability and transmembrane transport mechanism of drug molecule, has become research drug absorption feature Representative in vitro study system.
Kidney is one of first target organs of poisonous side effect of medicine.Kidney is by having different ultra microstructures, suction more than 20 kinds The cellularity of receipts ability and transport function, is the vitals of excretion of drug.Chemicals is through meeting selectivity during kidney excretion Ground is enriched in nephrocyte, causes cell membrane, mitochondria, endoplasmic reticulum and lyase bulk damage, is destroyed the integrality of cell, is made kidney thin Born of the same parents' apoptosis or necrosis.The filtration of glomerulus and the reabsorption function of renal tubule are reduced, in turn results in internal water and electrolyte Imbalance, acute renal failure can be also caused when serious.Therefore, observation medicine renal toxicity is Drug safety assessment and medicine poison The important content of pharmacological research.Modern medicines toxicologic study starts to be combined hair from In vivo study to internal and external research Exhibition, using internal and external technology, in the kidney of entirety, organ, cell, subcellular fraction and molecular water equality many levels research medicine Toxicity.In the in vitro study of medicine renal toxicity, establish kidney external model and be applied to the evaluation of vitro Drug renal toxicity Progressively become hot spot with screening.Work on hand mostly concentrates on medicine directly to the toxicity assessment of kidney, does not consider in medicine body Real process, as can the structure intestines-absorption process of kidney modeling medicine in vivo is of great significance to assess renal toxicity.
Microfluidic chip technology is presented as a science and technology developed rapidly in biomedical sector Its unique advantage, more because of it with cell size matching, environment is close with physiological environment, can carry on time and Spatial Dimension For more accurate manipulation, the features such as being easy to realize various kinds of cell functional study by flexible design, grinds as cell of new generation The Important Platform studied carefully.Having functional glomerulus chip using microflow control technique structure has highly important advantage and meaning.
At present, the complicated multiple organ chip for tool function being built using microflow control technique carries out correlative study analysis also in sky In the white stage, build using microflow control technique and combine the glomerulus chip with filtering function with reference to the intestines chip with absorption function With highly important advantage and meaning.As can realize that there is great application prospect in biological study and medicine research and development.
The content of the invention
In view of the above-mentioned problems, a kind of intestines of the aids drug body absorption process based on micro-fluidic chip of present invention offer- Kidney system, this method can carry out drug toxicity evaluation with absorption process in analogue body, applied to renal toxicity of the medicine after absorption Evaluation.
A kind of fluidic chip, the micro-fluidic chip are mainly made of top layer chip, porous membrane, bottom chip, porous filter For film by the lower surface of irreversible sealing-in top layer chip, the porous membrane lower surface that sealing has top layer chip passes through PDMS and bottom core The upper surface bonding sealing-in of piece；
Top layer chip is formed by connecting by the top layer chip main channel and top layer chip main channel inlets of U-shaped,
Bottom chip is by left side main channel, right side main channel and collagen passage, collagen feeder connection, bottom chip left side Main channel inlets composition on the right side of main channel inlets and bottom chip, the left even left side main channel of collagen passage, right even right side are main logical Road；
Top layer chip main channel is by connecting main channel on the left of bottom chip under porous membrane；Main channel leads on the left of bottom chip Cross on porous membrane and connect top layer chip main channel, right even collagen passage.
The left side main channel of the bottom chip is I shapes, and the right side main channel of the bottom chip is U-shaped, the bottom Chip cell collagen passage is " rich " font, is transversary on the position among collagen passage, and collagen passage passes through transverse direction Structure is connected with left channel and right channel, and the quantity of transversary is 1~10；Collagen passage and left side main channel It is concave surface to have a common boundary for hemispherical interface, collagen side.
The bottom chip is made of the different two parts of height, and left side main channel, right side main passage height are 200- 500 μm, collagen channel height is 80-200 μm, main passage height：Collagen channel height is 1~3:1.
The chip material is the PDMS polymer that light-permeable is breathed freely, and PDMS monomers are 5~10 with ratio of initiator:1, it is more Hole filter membrane material is polycarbonate membrane, and the aperture of polycarbonate membrane is 0.01~10um.
Main channel is designed as curved on the right side of top layer chip main channel and bottom chip, main channel and collagen on the left of bottom chip Passage is designed as straight type, in this way, each feeder connection can be avoided mutually, and have certain distance, does not interfere with operation, avoids distance Cross nearly caused contact stain.The lower surface of every layer of chip and porous membrane are irreversible sealing-in, the upper surface of every layer of chip and Porous membrane bonds for PDMS.
The preparation method of micro-fluidic chip provided by the invention, when irreversible method for sealing is that UV activation 1 is small, silanization 30 points of processing, oxygen plasma sealing-in.
It using monomer and ratio of initiator is 20 that PDMS adhesive bonding methods, which are,:1 PDMS polymer, get rid of 10 on slide~ 50um is thick, and after chip upper surface dips PDMS, sealing-in of aliging with the irreversible porous membrane for being sealed with upper strata chip, is put into 80 Degree baking oven, 30 points.
A kind of intestines-kidney system of the aids drug body absorption process based on micro-fluidic chip, using above-mentioned micro-fluidic core Piece, it is built-up in accordance with the following methods：
(1) chip pre-processes
Chip is designed and produced, prepared collagen working solution is added into collagen passage with pipettor, adds 1mL PBS buffer In culture dish, the culture dish of fixed chip is put into incubator and is incubated 30min, promotes collagen to be changed into fruit from viscous liquid Freeze shape gel, after gel process, cell main channel adds fresh cell culture fluid；
(2) inoculation and culture of cell
Enterocyte is adjusted to 1~5 × 106The cell suspension of cells/mL, add upper strata chip main channel, cell attachment in Porous membrane interface growth, chip translation is put into 37 DEG C of incubators and continues culture, change liquid once every 24h；
Primary rat glomerulus micro-assembly robot is extracted, is adjusted to 104~105The cell suspension of cells/mL, takes 10 μ L cells to hang Liquid adds left side main channel, and left side main channel is upward, and collagen passage edge-on overnight incubation downwards, makes cell attachment in bottom chip Left side main channel and the hemispherical interface of collagen passage, observe visible glomerulus micro-assembly robot and are attached at collagen under an optical microscope Interface growth, chip translation is put into 37 DEG C of incubators and continues culture, change liquid once every 24h；
(3) treat that cell growth state is good, follow-up test can be carried out；Medicine is observed after absorption to the shadow of glomerular function Ring and other.
A kind of application of intestines-kidney chip of aids drug body absorption process using above method structure, its feature exist In：Above-mentioned system can be used, drug toxicity is evaluated, detailed process is as follows：
(1) detection of cytoactive, CCK-8 reagents are carried out using CCK-8 kits：The volume ratio of cell culture medium is 1: 9, be mixed into detection working solution, add main channel on the left of bottom chip, put in incubator 3 it is small when after, be transferred in 96 orifice plates, Absorbance at microplate reader measure 450nm, for being quantified to cell viability, one of index as drug toxicity；
(2) LDH is leaked out, and collects chip channel inner cell nutrient solution, and using LDH kits, operation detection LDH concentration, is used One of degree of injury in observation medicine to cell, index as drug toxicity；
(3) immunofluorescence, routine immunization dying operation, fluorescence microscope are taken pictures, and carry out Live/Dead dyeing, are seen Examine cell viability；ZO-1 is dyed, and observes cell tight junction situation；F-actin is dyed, and observation cytoskeletal protein etc. is used as medicine The index of thing toxicity；
(4) albumen filters, and uses albumin (MW:70kDa), add left side main channel, 1 it is small when after, it is main logical to collect right side Road nutrient solution, albumin reagent box operation detection protein concentration, the filtration rate of observation middle-molecular-weihydroxyethyl albumin, as medicine to kidney The index that filtering function influences；
(5) albumen filters, and uses Igg (MW:150kDa), add left side main channel, under fluorescence microscope in 0min, 15min, 30min, 60min take pictures, and analyze Igg permeability, observe the filtration rate of high-molecular-weight protein, and kidney is filtered as medicine The index of function effect.
Intestines-kidney system of aids drug body absorption process of the invention based on micro-fluidic chip, top layer chip are inoculated with Enterocyte, can be with the absorption process of medicine in analogue body；Bottom chip mainly passes through glue by left side main channel and right side main channel Former passage connection.Glomerulus micro-assembly robot is inoculated with left side main channel, glomerulus micro-assembly robot is led in left side master by edge-on culture The hemispherical interface in road and collagen passage grows and forms barrier cell, and it is small can to observe kidney as collecting region for right side main channel Influence of the renal toxicity to detection of glomeruli filtration function caused by the filtration of ball, and chemical composition of the medicine after intestinal absorption. Realize the structure of the drug toxicity appraisement system of absorption process in analogue body, and its evaluate and answer in renal toxicity of the medicine after absorption With.
Intestines-kidney system of aids drug body absorption process of the invention based on micro-fluidic chip, constructing one can The renal toxicity evaluation platform of absorption process in analogue body, is not only able to the absorption of aids drug in vivo, observes the mutual of medicine The influence to absorption is acted on, while can realize evaluation kidney filtering function in vitro.Drug evaluation is carried out with this platform, acquisition Data more meet the actual conditions of people, reduce because the medicament research and development later stage caused by research and development drug toxicity data early period are unreliable Mortality.
Brief description of the drawings
Fig. 1 micro-fluidic chip schematic diagrames of the present invention；A bottom chip collagen channel design schematic diagrames；B bottom chips main channel Structure diagram；C bottom chip schematic top plan views；D bottom chip collagens interface enlarged diagram；E top layer liver chip structures show It is intended to；F invention micro-fluidic chip schematic top plan views；G invention micro-fluidic chip overall schematics；H invention micro-fluidic chips are transversal Face schematic diagram.
Wherein：1 top layer chip main channel, 2 enterocytes, 3 bottom chips left side main channel, 4 glomerulus micro-assembly robots, 5 is porous Filter membrane, 6 bottom chips right side main channel, 7 collagen passages, 8 collagen feeder connections, 9 bottom chips left side main channel inlets, 10 bottoms Main channel inlets on the right side of layer chip, 11 top layer chip main channel inlets, 12 hemispherical interfaces.
Fig. 2 intestines of the present invention-kidney chip gut function exterior syndrome；A intestinal villus structures；B intestinal villus polarity, alkaline phosphatase are quantitative；c Enterocyte tight junction protein (ZO-1) immunofluorescence dyeing.
The renal toxicity evaluation of Fig. 3 medicines；The Live/Dead colored graph b glomerulus CCK-8 vigor quantitative results of a glomerulus； The influence that c medicines leak out glomerulus LDH.
Influence of Fig. 4 medicines to detection of glomeruli filtration function；Permeability of a Igg in glomerulus；B albumin is in glomerulus Permeate quantitative result.
Embodiment
The following examples will be further described the present invention, but not thereby limiting the invention.
Embodiment 1
Design and make micro-fluidic chip, construction is as shown in Figure 1.The micro-fluidic chip is mainly by top layer chip, porous filter Film, bottom chip composition, porous membrane seal the porous membrane for having top layer chip by the lower surface of irreversible sealing-in top layer chip Lower surface bonds sealing-in by the upper surface of PDMS and bottom chip；
Top layer chip is formed by connecting by the top layer chip main channel 1 and top layer chip main channel inlets 11 of U-shaped；
Bottom chip is left by left side main channel 3, right side main channel 6, collagen passage 7, collagen feeder connection 8, bottom chip Main channel inlets 10 form on the right side of side main channel inlets 9 and bottom chip, the left even left side main channel 3 of collagen passage 7, right even right Side main channel 6；
Top layer chip main channel 1 is by connecting main channel 3 on the left of bottom chip under porous membrane 5；It is main logical on the left of bottom chip Road 3 is by connecting top layer chip main channel 1, right even collagen passage 7 on porous membrane 5.
The left side main channel 3 of the bottom chip is I shapes, and the right side main channel 6 of the bottom chip is U-shaped, the bottom Layer chip cell collagen passage 7 is " rich " font, is transversary on the position among collagen passage, collagen passage passes through horizontal stroke It is connected to structure with left channel 3 and right channel 6, the quantity of transversary is 3；Collagen passage 7 and left side main channel 3 Boundary be hemispherical interface 12, collagen side is concave surface.
The bottom chip is made of the different two parts of height, and left side main channel 3,6 height of right side main channel are 300 μm, 7 height of collagen passage is 100 μm, main passage height：Collagen channel height is 3:1.
The chip material is the PDMS polymer that light-permeable is breathed freely, and PDMS monomers are 5 with ratio of initiator:1, porous filter Membrane material is polycarbonate membrane, and the aperture of polycarbonate membrane is 0.4um.
Compound concentration is the collagen working solution of 4mg/mL, by collagen perfusion to chip collagen passage, after incubation 30min, respectively Chip channel adds cell culture fluid.Enterocyte is adjusted to suitable cell density, adds upper strata chip main channel, cell attachment In porous membrane interface growth, chip translation is put into 37 DEG C of incubators and continues to cultivate.After enterocyte culture 14 days, extraction is former For Renal Glomeruli In Rats micro-assembly robot, adjust to suitable cell density, take 10 μ L cell suspensions to add main channel on the left of lower layer chip, Edge-on overnight incubation, observes visible glomerulus micro-assembly robot and is attached at collagen interface growth, chip is translated under an optical microscope It is put into 37 DEG C of incubators and continues culture, changes liquid once every 24h.
Scanning electron microscopic observation enterocyte form, as shown in Figure 2 a, it is seen that obvious intestinal villus structure.Collect upper strata chip (AP) nutrient solution and lower layer chip (BL) nutrient solution detection of alkaline phosphatase activity, as a result as shown in Figure 2 b, it is seen that ap layers of alkalescence Phosphatase activity is the mark of intestinal villus polarity apparently higher than bp layers.ZO-1 immunofluorescence dyeings are carried out, as shown in Figure 2 c, can Seeing has obvious tight connecting device.Intestines chip has complete individual layer barrier function.
Embodiment 2
The evaluation application of medicine renal toxicity
Digoxin has renal toxicity, and Cholestyramine can reduce the intestinal absorption of digoxin, and Verapamil then increases digoxin Intestinal absorption, renal toxicity changes therewith.
Build intestines-kidney chip.Upper strata chip channel is separately added into containing 80 μM of digoxin (Dig), 80 μM of connection of digoxin (Dig) Conjunction Cholestyramine (Col) 200 μ g/mL, the cell culture fluid cultures of 80 μM of 20 μM of joint Verapamils (Ver) of digoxin (Dig), Handle 48 it is small when after collect lower layer chip main channel nutrient solution and carry out LDH and leak out detection, as a result as shown in Figure 2 c.As it can be seen that ground is high Pungent combination Cholestyramine leaks out low compared with the LDH that digoxin is used alone；Digoxin is relatively used alone in digoxin combination Verapamil LDH leak out height, drug combination have impact on the renal toxicity of digoxin after enterocyte absorbs.CCK-8 is carried out to glomerulus to quantify Test and life or death are dyed, and fluorescence microscope is taken pictures, as a result as shown in Figure 2 a and 2 b.Digoxin combination Verapamil more individually should More with the dead cell quantity of digoxin, cell viability substantially reduces, and digoxin is relatively used alone in digoxin combination Cholestyramine Dead cell quantity it is few, cell viability increases, and renal toxicity of the drug combination after intestinal absorption is changed.It is left in lower layer chip Side main channel is separately added into Igg (red fluorescence), takes pictures under fluorescence microscope in 0min, 15min, 30min, 60min.As a result such as Shown in Fig. 3 a, the Igg that molecular weight is 150000 is more in Verapamil combination digoxin in the infiltration of glomerulus chip, and comes examining Enamine combination digoxin is less.Albumin (molecular weight 70000) titer 15mg/mL add left side main channel, 1 it is small when after, receive Collection right side main channel nutrient solution, operates detection protein concentration, as a result as shown in Figure 3b using albumin reagent box.It can be seen that medicine joins The detection of glomeruli filtration function of renal toxicity index is used as by the use of enteral inhalation effects.

Claims (6)

  1. A kind of 1. micro-fluidic chip, it is characterised in that：The micro-fluidic chip is mainly by top layer chip, porous membrane, bottom chip Composition, by the lower surface of irreversible sealing-in top layer chip, the porous membrane lower surface that sealing has top layer chip passes through porous membrane The upper surface of PDMS and bottom chip bonds sealing-in；
    Top layer chip is formed by connecting by the top layer chip main channel (1) and top layer chip main channel inlets (11) of U-shaped；
    Bottom chip is by left side main channel (3), right side main channel (6), collagen passage (7), collagen feeder connection (8), bottom core Main channel inlets (9) and bottom chip right side main channel inlets (10) composition on the left of piece, the left even left side of collagen passage (7) is main to be led to Road (3), right even right side main channel (6)；
    Top layer chip main channel (1) is by connecting main channel (3) on the left of bottom chip under porous membrane (5)；It is main on the left of bottom chip Passage (3) is by connecting top layer chip main channel (1), right even collagen passage (7) on porous membrane (5).
  2. 2. micro-fluidic chip described in accordance with the claim 1, it is characterised in that：The left side main channel (3) of the bottom chip is I Shape, the right side main channel (6) of the bottom chip is U-shaped, and the bottom chip cell collagen passage (7) is " rich " font, It is transversary on position among collagen passage, collagen passage passes through transversary and left channel (3) and right channel (6) It is connected, the quantity of transversary is 1~10；Collagen passage (7) and the boundary of left side main channel (3) are hemispherical interface (12), collagen side is concave surface.
  3. 3. micro-fluidic chip described in accordance with the claim 1, it is characterised in that：The bottom chip is by different two of height It is grouped into, left side main channel (3), right side main channel (5) they are highly 200-500 μm, and collagen passage (6) is highly 80-200 μm, Main passage height：Collagen channel height is 1~3:1.
  4. 4. micro-fluidic chip described in accordance with the claim 1, it is characterised in that：The chip material is the PDMS that light-permeable is breathed freely Polymer, PDMS monomers are 5~10 with ratio of initiator:1, porous membrane material is polycarbonate membrane, the hole of polycarbonate membrane Footpath is 0.01~10um.
  5. A kind of 5. intestines-kidney system of the aids drug body absorption process based on micro-fluidic chip, it is characterised in that：Using above-mentioned Micro-fluidic chip, it is built-up in accordance with the following methods：
    (1) chip pre-processes
    Chip is designed and produced, prepared collagen working solution is added into collagen passage with pipettor, adds 1mL PBS buffer in training Support in ware, the culture dish of fixed chip is put into incubator and is incubated 30min, promotes collagen to be changed into g., jelly-like from viscous liquid Gel, after gel process, cell main channel adds fresh cell culture fluid；
    (2) inoculation and culture of cell
    Enterocyte is adjusted to 1~5 × 106The cell suspension of cells/mL, adds upper strata chip main channel, cell attachment is in porous Filter membrane interface growth, chip translation is put into 37 DEG C of incubators and continues culture, change liquid once every 24h；
    Primary rat glomerulus micro-assembly robot is extracted, is adjusted to 104~105The cell suspension of cells/mL, takes 10 μ L cell suspensions to add Enter left side main channel, left side main channel is upward, and collagen passage edge-on overnight incubation downwards, makes cell attachment on the left of bottom chip Main channel and the hemispherical interface (12) of collagen passage, observe visible glomerulus micro-assembly robot and are attached at collagen under an optical microscope Interface growth, chip translation is put into 37 DEG C of incubators and continues culture, change liquid once every 24h；
    (3) treat that cell growth state is good, follow-up test can be carried out；Observe influence of the medicine after absorption to glomerular function and Other.
  6. 6. should according to a kind of intestines-kidney system of the aids drug body absorption process based on micro-fluidic chip described in claim 5 With, it is characterised in that：Above-mentioned system can be used, drug toxicity is evaluated, detailed process is as follows：
    (1) detection of cytoactive, CCK-8 reagents are carried out using CCK-8 kits：The volume ratio of cell culture medium is 1:9, mix Close uniformly into detection working solution, add main channel (3) on the left of bottom chip, put in incubator 3 it is small when after, be transferred in 96 orifice plates, Absorbance at microplate reader measure 450nm, for being quantified to cell viability, one of index as drug toxicity；
    (2) lactic dehydrogenase (LDH) leaks out, and collects chip channel inner cell nutrient solution, uses LDH kits, operation detection training Nutrient solution LDH leaks out concentration, for observing one of degree of injury of the medicine to cell, index as drug toxicity；
    (3) immunofluorescence, routine immunization dying operation, fluorescence microscope are taken pictures, and carry out Live/Dead dyeing, and observation is dead Viable count；ZO-1 is dyed, and observes cell tight junction situation；F-actin is dyed, and observation cytoskeletal protein etc. is used as medicine The index of toxicity；
    (4) albumen filters, and using albumin, adds left side main channel, 1 it is small when after, collect right side main channel nutrient solution, albumin Kit operation detection protein concentration, observes the filtration rate of middle-molecular-weihydroxyethyl albumin, kidney filtering function is influenced as medicine Index；
    (5) albumen filters, and using Igg, adds left side main channel, is clapped under fluorescence microscope in 0min, 15min, 30min, 60min According to analyzing Igg permeability, observe the filtration rate of high-molecular-weight protein, the index influenced as medicine on kidney filtering function.
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