CN106566801A - Method for establishing osteoporosis model based on microfluidic technology - Google Patents
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Abstract
The invention provides a method for establishing an osteoporosis model based on the microfluidic technology. The method comprises the following steps: manufacturing a material by adopting polydimethylsiloxane (PDMS) and glass as core plates, selecting the mouse embryo osteoblast MC3T3-E1 cell line as basic cells, carrying out osteogenesis differentiation induction on the basic cells, carrying out perfusion treatment of hexadecadrol culture solution (with the concentration of 10 mu mol/L) on the differentiation induced osteoblasts in the core plates for 24h at the speed of 0.1 mul/min by adopting a perfusion device, thus establishing an osteoporosis case simultaneous model; on the other hand, carrying out perfusion treatment of hexadecadrol and lithium chloride medicine (with the concentration of 25 mmol/L) on the differentiation induced osteoblasts in the core plates for 24h at the speed of 0.1 mul/min by adopting the perfusion device, thus establishing an osteoporosis treatment module. With the model established by adopting the method provided by the invention, a potential application platform is provided for the fields of tissue engineering, regenerative medicine and the like, and meanwhile, the operation is simple and flexible, the controllability is strong, the automation degree is high, and the consumed amount of the reagent is small.
Description
Technical field
The invention belongs to microfluidic chip technology, tissue engineering technique, regenerative medicine and its cytological applications etc.
A kind of field, and in particular to method that osteoporosis model is set up based on microflow control technique.
Background technology
Osteoporosises are a kind of multi-pathogenesis onsets, and bone amount is reduced with unit volume, bone micro-structure is destroyed as spy
The common orthopaedics metabolic disease of point, is clinically generally increased with skeleton pain, bone fragility, easily sends out fracture
It is characterized.Osteoporosis can be divided into constitutional, Secondary cases and idiopathic three major types.Primary osteoporosis
Disease accounts for the 90% of osteoporosis, is divided into I type and II type.I type is postmenopausal osteoporosiss
(post-menopausal osteoporosis, PMOP), II type is senile osteoporosises (senile
Osteoporosis, SOP) (Cheng Xiaozhi, Yan Dong. the imaging diagnosises of osteoporosis. new medical science, 2007,
38(1):11-13).In life, bone is in continuous renewal state to people, and osteoclast removes old bone (bone resorption),
It is followed by osteoblast and fills up new bone (bone formation), the referred to as reconstruction of bone.And the bone for working as Osteoclasts mediate is inhaled
Adduction it is absolute or relative then occur that bone amount is persistently lost and bone is close when increasing above osteoblastic bone formation
The continuous decrease of degree be osteoporosises occur (He Wei's great waves, Liu Kang, Sun Jin are all, etc. cathepsin K and
The progress of osteoporosis treatment. Chinese osteoporosises magazine, 2008,14 (9):671).The whole world is about
There is population more than 200,000,000 with osteoporosises, only just have 88,000,000 in China, wherein middle-aged and elderly people, post menopausal female
Property accounts for the overwhelming majority.And increasing with aged tendency of population, osteoporosises will have a strong impact on the life of people
Health living.Past 10 years, osteoporotic pathological process was contacted with tissue, cell and molecular level
Together.As signal integration various endocrine, neuroendocrine, inflammatory and the mechanical force of " main switch "
Stimulation.In cellular level, main osteocyte (bone formation osteoblast, bone degraded osteoclast)
Interaction and coupling define the minimum functional unit of bone, and have some important molecules in bone remoulding mistake
Coordinate the activity between osteoblast and osteoclast in journey.Osteoclast, originate from hematopoietic stem cell and with
Monocyte-macrophage is closely related.Osteoblast, represents a class by mesenchymal stem cells differentiation
Bone formation cell.Speed and effect of its precursor to ripe osteoblast (secretion can mineralized dentin matrix) differentiation
Rate and osteoblastic life-span determine osteoplastic speed.At present, for osteoporotic treatment still lacks
Weary effective external model is furtheing investigate its pathomechanism, while more lacking the scheme of effectively treatment.
Microfluidic chip technology is familiar rapidly in recent years, and to biomedical sector infiltration,
Its wide application prospect is highlighted, increasing researcher is using microfluidic chip technology as biomedicine
The important experiment porch of research, in also gradually penetrating into the research of some clinics.Micro-fluidic chip is also known as core
Piece laboratory, refers to one several square centimeters of chip, is tested in biology, chemistry etc. with its microscale spatial
It is middle by the functional new science and technology being integrated on the chip.The appearance of microfluidic chip technology,
The new important technological platform being considered required for cell research.Its main feature and advantage is that energy will be various
Monotechnics carries out integration, with controllability.Biomedical research can in practical study and demand,
With reference to basis and clinical research, related micro fluidic chip is designed and prepared, the development of this technology can be brought into one
Individual new field, can also make some cannot break through in biomedical research a difficult problem in the platform of micro-fluidic chip
Under obtain breakthrough progress.
The content of the invention
It is an object of the invention to provide a kind of method for setting up osteoporosis model based on microflow control technique, the party
Method solve present in conventional external structure osteoporosis model process drug concentration gradient generate it is unstable,
The problems such as poor controllability.
The invention provides a kind of method that osteoporosis model side is set up based on microflow control technique, the tool of the method
Body step is as follows：
--- with SU8 photoresists as template, made with tree-like passage and cell culture chamber with soft lithography
Micro-fluidic chip；Micro-fluidic chip is formed by the PDMS on upper strata and the sheet glass sealing-in of lower floor, with solution
Inlet port, Concentraton gradient production unit, cell culture chamber, waste liquid port and cell inoculation mouth；Solution inlet port
Connection Concentraton gradient production unit, afterwards with cell culture chamber UNICOM, cell culture chamber respectively with waste liquid port and carefully
Born of the same parents' inoculation mouth UNICOM；
--- the osteoblast of induction passes through cell inoculation mouth and passage to cell culture chamber, adherent rear for real
Test；
--- two syringes with pump are connected at the solution inlet port of said chip, syringe is built-in
Complete culture solution and concentration are 10 μm of ol/L culture fluid containing dexamethasone, are irrigated simultaneously by solution inlet port
24h, speed is 0.1 μ l/min, and liquid is filled in after Concentraton gradient forming part in six cell culture chambers
The concentration of meter Song is by respectively 0,2,4,6,8,10 μm of ol/L；
--- after chip inner cell it is adherent, propagation after, by concentration be 25mmol/L lithium chlorides inject cell；
Pretreatment 1h is carried out to cell in cell culture room, then by chloride containing lithium culture fluid and containing dexamethasone and
The culture fluid of lithium chloride irrigates 24h simultaneously by solution inlet port respectively, and speed is 0.1 μ l/min；
--- activity and propagation of the MC3T3-E1 osteoblast in chip is investigated, and does statistical analysiss.
The micro-fluidic chip has 6 parallel cell culture chambers, and the size of each cell culture chamber is：
Long 1200 μm, wide 1200 μm, high 150 μm, volume is about 2 μ L, and is inoculated with independent cell
Mouthful.
Described micro-fluidic is PDMS and glass.
Osteoporosises pathology and treatment model are built using dexamethasone culture fluid and lithium chloride solution.
The structure of the tree-like passage of the micro-fluidic chip can be according to the solution concentration of two injection ports by multiple
Fluid distribution forms concentration.
Due to the motility of microflow control technique, this method can according to solution inlet port solution type, characteristic,
Concentration designs the methods such as multistage hybrid channel, so as to realize the drug concentration gradient of complexity.Cell inoculation mouth can
Inoculating cell suspension or the cell mixture containing extracellular matrix are cultivated realizing cell 2D and 3D.
The present invention provides microflow control technique and sets up osteoporotic model method, is using dexamethasone culture fluid
Osteoporotic pathology and treatment model are built with lithium chloride solution.The concentration of dexamethasone medicine is
10 μm of ol/L, the concentration of lithium chloride medicine is 25mmol/L.24h is irrigated by solution inlet port simultaneously, speed
Spend for 0.1 μ l/min.
The present invention using micro-fluidic chip as main technology platforms, with cells in vitro structure based on osteoblast
The osteoporosis model (GIOP) of hormone induction is built, and different phase sclerotin is simulated according to the characteristics of chip and dredged
There is development and pass through and it is treated in the state of pine, observation osteoporosises.
In sum, the invention provides the strong external structure sclerotin of a kind of effective, flexible operation, controllability
Loose method, and method of the generation with dexamethasone gradient that can be realized with a step, with very heavy
Want meaning.
The offer microflow control technique that the present invention is provided sets up osteoporotic model method, has an advantage in that
1st, the drug level of gradient can be produced by one-step method；
2nd, the controllability of medicine Gradient distribution is strong；
3 and cell culture chamber it is integrated, cell two and three dimensions culture can be carried out；
4th, simple to operate, quick, flexible and high degree of automation；
5th, can be by integrated raising flux；
6th, reagent consumption is little.
Description of the drawings
Fig. 1 is the micro-fluidic chip schematic diagram that osteoporosis model method is set up based on microflow control technique；
Fig. 2 is that the microfluidic chip structure details for setting up osteoporosis model method based on microflow control technique is illustrated
Figure；
The Concentraton gradient production unit of 1,2 solution inlet port of wherein 1 solution inlet port 2,3,4 cell culture chambers,
5 waste liquid ports, 6 cell inoculation mouths；
Fig. 3 is to set up the Concentraton gradient Fluorescent Characterization that osteoporotic model method is produced based on microflow control technique
Figure；
Fig. 4 is that MC3T3-E1 cell drugs process (dexamethasone and lithium chloride) 24 on micro-fluidic chip
Cell propagation statistics after hour；
Fig. 5 is that MC3T3-E1 cell drugs process (dexamethasone and lithium chloride) 24 on micro-fluidic chip
Apoptosis statistics after hour；
Fig. 6 is the detection of osteoblast alkaline phosphatase activitieses.
Specific embodiment
Following examples will be further described to the present invention, but not thereby limiting the invention.
Embodiment 1
1. the making of the micro-fluidic chip of polymer mixed.The negative glue pattern plates of SU8 are made using traditional soft lithography
And the PDMS block (monomers with passage accordingly are obtained by perfusion：Initiator=10：1).Chip is total to
There is upper and lower two-layer：Micro-fluidic chip is formed by the PDMS on upper strata and the sheet glass sealing-in of lower floor, and upper strata is logical
Channel layer, lower floor is blank glass piece, as shown in Figure 1.With solution inlet port 1 and solution inlet port 2, dense
Degree gradient production unit 3, cell culture chamber 4, waste liquid port 5 and cell inoculation mouth 6；Solution inlet port connects
Concentraton gradient production unit 3, afterwards with the UNICOM of cell culture chamber 4, cell culture chamber 4 respectively with waste liquid port 5 and
Cell inoculation mouth UNICOM 6；
When two kinds of liquid are descending along Concentraton gradient passage production unit 3, every kind of liquid all will be punished in node
Then stream merged with the liquid stream of surrounding in the way of laminar flow, is then mixed (such as Fig. 2 institutes in sinuous passage
Show).Liquid continuous dilution, finally forms a series of concentration in Concentraton gradient delivery outlet, and concentration is characterized as schemed
Shown in 3.According to studying at this stage, if the strength of fluid of two inlet openings is respectively 0 and C, this six outputs
The concentration of mouth will be respectively 0,1/5C, 2/5C, 3/5C, 4/5C and C.Cell culture part is by six cultures
Pond constitutes and in combination with Concentraton gradient forming part, and the size of each culture pond is：It is long 1200 μm, it is wide
1200 μm, high 150 μm, volume is about 2 μ l.Osteoblast reaches culture pond by inlet opening and passage,
It is adherent after overnight.
Inoculation of 2 osteoblast in micro-fluidic chip and culture
Chip after sealing-in is placed in the culture dish of 25mm diameters, is rinsed one time with PBS and then is used again
DMEM culture fluid rinse one time, to guarantee that bubble-free is present in passage.By the postdigestive osteoblast of pancreatin
By Cell infusion hole with 4 × 105The even density of/ml is inoculated on chip, is subsequently placed into incubator overnight incubation.
The foundation of 3 osteoporosis models
It is respectively that complete culture solution and culture fluid containing dexamethasone is (dense after Microscopic observation cell attachment, propagation
Spend for 10 μm of ol/L) pass through two solution injection ports while irrigating 24h, speed is 0.1 μ l/min (this speed stream
Body shearing force will not be to affecting cells), liquid after Concentraton gradient forming part, six cell culture
The concentration of indoor dexamethasone is by respectively 0,2,4,6,8,10 μm of ol/L.
The treatment of 4 osteoporosis models
After chip inner cell it is adherent, propagation after, by lithium chloride (concentration is 25mmol/L) inject cell culture
Pretreatment 1h is carried out to cell in pond, then by chloride containing lithium culture fluid and containing dexamethasone and lithium chloride
Culture fluid irrigates 24h simultaneously by solution inlet port respectively, and speed is 0.1 μ l/min.
Embodiment 2
Dexamethasone and lithium chloride act on the impact to MC3T3-E1 activity and propagation simultaneously
To the MC3T3-E1 lithium chloride pretreatment 1h after induction, lithium chloride is then carried out to it and dexamethasone is filled
After stream 24h, the osteoblast in six cell culture chambers is in form, multiplication rate and activity without substantially poor
It is different, as shown in Figure 4 and Figure 5, illustrate what cell propagation that lithium chloride causes to dexamethasone and activity were reduced
Affect that there is therapeutical effect.Dexamethasone in osteoblasts is recorded by the detection to apoptosis marker to wither
Die the impact of damage, MMP reflection early stages reversible apoptosis is damaged, and the change of nuclear morphology and plasma membrane permeability
Then represent cell injury late period irreversible apoptosis to damage.The correlation analysiss of these indexs can reflect difference
The state of apoptosis phase cell, so as to reflect process of the medicine to cell injury.
Embodiment 3
Osteoblast alkaline phosphatase activitieses are detected
MC3T3-E1 cells after induction Jing dexamethasone culture fluid perfusion 24h process after, Cellular alkaline phosphatase
Activity compared with significantly decreasing before perfusion, with obvious significant difference, illustrate that dexamethasone can be bright
Osteoblastic alkaline phosphatase activitieses are affected aobviously；MC3T3-E1 cell Jing lithium chloride perfusions after induction
After 24h process, the activity of Cellular alkaline phosphatase changes compared with before perfusion without obvious, as shown in fig. 6, explanation
Lithium chloride has no significant effect to the activity of osteoblast alkali phosphatase；To the MC3T3-E1 chlorinations after induction
Lithium pretreatment 1h, after lithium chloride and dexamethasone perfusion 24h are carried out to it, Cellular alkaline phosphatase activity compared with
Without significant change before perfusion, illustrate that the Cellular alkaline phosphatase activity that lithium chloride is caused to dexamethasone reduces tool
There is therapeutical effect.
Claims (5)
1. a kind of method that osteoporosis model is set up based on microflow control technique, it is characterised in that：The method
Comprise the following steps that：
--- with SU8 photoresists as template, made with tree-like passage and cell culture chamber with soft lithography
Micro-fluidic chip；Micro-fluidic chip is formed by the PDMS on upper strata and the sheet glass sealing-in of lower floor, with solution
Inlet port, Concentraton gradient production unit, cell culture chamber, waste liquid port and cell inoculation mouth；Solution inlet port
Connection Concentraton gradient production unit, afterwards with cell culture chamber UNICOM, cell culture chamber respectively with waste liquid port and carefully
Born of the same parents' inoculation mouth UNICOM；
--- the osteoblast of induction passes through cell inoculation mouth and passage to cell culture chamber, adherent rear for real
Test；
--- two syringes with pump are connected at the solution inlet port of said chip, syringe is built-in
Complete culture solution and concentration are 10 μm of ol/L culture fluid containing dexamethasone, are irrigated simultaneously by solution inlet port
24h, speed is 0.1 μ l/min, and liquid is filled in after Concentraton gradient forming part in six cell culture chambers
The concentration of meter Song is by respectively 0,2,4,6,8,10 μm of ol/L；
--- after chip inner cell it is adherent, propagation after, by concentration be 25mmol/L lithium chlorides inject cell；
Pretreatment 1h is carried out to cell in cell culture room, then by chloride containing lithium culture fluid and containing dexamethasone and
The culture fluid of lithium chloride irrigates 24h simultaneously by solution inlet port respectively, and speed is 0.1 μ l/min；
--- activity and propagation of the MC3T3-E1 osteoblast in chip is investigated, and does statistical analysiss.
2., according to the method for setting up osteoporosis model based on microflow control technique described in claim 1, it is special
Levy and be：The micro-fluidic chip has 6 parallel cell culture chambers, and the size of each little culturing room is：
Long 1200 μm, wide 1200 μm, high 150 μm, volume is about 2 μ L, and with independent cell inoculation mouth.
3., according to the method for setting up osteoporosis model based on microflow control technique described in claim 1, it is special
Levy and be：Described micro-fluidic is PDMS and glass.
4., according to the method for setting up osteoporosis model based on microflow control technique described in claim 1, it is special
Levy and be：Osteoporosises pathology and treatment model are built using dexamethasone culture fluid and lithium chloride solution.
5., according to the method for setting up osteoporosis model based on microflow control technique described in claim 1, it is special
Levy and be：The structure of the tree-like passage of the micro-fluidic chip can pass through according to the solution concentration of two injection ports
Repeatedly fluid distribution forms concentration.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117511739A (en) * | 2024-01-04 | 2024-02-06 | 中日友好医院(中日友好临床医学研究所) | Construction method and device of microfluidic bone organ chip |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102978109A (en) * | 2012-11-06 | 2013-03-20 | 中国科学院大连化学物理研究所 | Establishment and characterization method of in-vitro blood brain barrier model based on microfluidic chip |
| CN103173871A (en) * | 2011-12-22 | 2013-06-26 | 中国科学院大连化学物理研究所 | Method for producing nano electrospining with concentration gradient based on microfluidics technology |
| CN103789206A (en) * | 2014-02-18 | 2014-05-14 | 中国科学院电子学研究所 | Osteoblast electrical stimulation system based on microfluidic technology and operation method thereof |
| CN104630059A (en) * | 2015-01-16 | 2015-05-20 | 中国科学院深圳先进技术研究院 | Microfluidic chip and method for establishing in-vitro co-culture model of three kinds of cells |
-
2015
- 2015-10-10 CN CN201510651915.3A patent/CN106566801B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173871A (en) * | 2011-12-22 | 2013-06-26 | 中国科学院大连化学物理研究所 | Method for producing nano electrospining with concentration gradient based on microfluidics technology |
| CN102978109A (en) * | 2012-11-06 | 2013-03-20 | 中国科学院大连化学物理研究所 | Establishment and characterization method of in-vitro blood brain barrier model based on microfluidic chip |
| CN103789206A (en) * | 2014-02-18 | 2014-05-14 | 中国科学院电子学研究所 | Osteoblast electrical stimulation system based on microfluidic technology and operation method thereof |
| CN104630059A (en) * | 2015-01-16 | 2015-05-20 | 中国科学院深圳先进技术研究院 | Microfluidic chip and method for establishing in-vitro co-culture model of three kinds of cells |
Non-Patent Citations (2)
| Title |
|---|
| WEILIANG YU, ET AL.: "A Mcirofluidic-Based Multi-Shear Device for Investigating the Effects of Low Fluid-Induced Stresses on Osteoblasts", 《PLOS ONE》 * |
| 李盈义: "基于微流控芯片骨质疏松模型的建立及治疗", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117511739A (en) * | 2024-01-04 | 2024-02-06 | 中日友好医院(中日友好临床医学研究所) | Construction method and device of microfluidic bone organ chip |
| CN117511739B (en) * | 2024-01-04 | 2024-03-12 | 中日友好医院(中日友好临床医学研究所) | Construction method and device of microfluidic bone organ chip |
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