CN1277920C - Device for making microencapsulation cell - Google Patents
Device for making microencapsulation cell Download PDFInfo
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- CN1277920C CN1277920C CN 03148551 CN03148551A CN1277920C CN 1277920 C CN1277920 C CN 1277920C CN 03148551 CN03148551 CN 03148551 CN 03148551 A CN03148551 A CN 03148551A CN 1277920 C CN1277920 C CN 1277920C
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- sodium alginate
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Abstract
The present invention relates to a device for producing encapsulization cells, which belongs to the field of biomedical engineering. The device is composed of an injection system, a high-voltage pulse generator and an auxiliary system. Microcapsules with different diameters from 50 to 1000 micrometers are produced by adjusting voltage, frequency and duty ratios. The produced microcapsules can be used for culturing cells and transplanting cells.
Description
Technical field
The invention belongs to biomedical engineering field, be specifically related to a kind of device for preparing micro-capsule, the micro-capsule of this device preparation can be used for bag by somatocyte, carries out cell cultures and Transplanted cells as islet cells, liver cell etc.
Background technology
Micro-capsule has selective permeability.It can stop the cells contacting of immunocyte, immunoglobulin (Ig) and bag quilt, thereby stops the repulsion of acceptor immunity system to graft.But micro-capsule allows the product of cellular metabolism desired nutritional material and cellular metabolism to pass through, and makes the transplanted cells can long-term surviving in micro-capsule and bring into play function.Good micro-capsule is except that must having certain permeability and immune buffer action, and excellent biological compatibility also is its characteristic that must have.Past people has been invented some fabrication techniques micro-capsules, for example the gas flow drop producer of resonator that uses in the United States Patent (USP) 4352883 and United States Patent (USP) 4391909 uses.But there are two shortcomings in the micro-capsule that above two kinds of methods are made: 1. volume is big, 700-1000 μ m; 2. shape is inhomogeneous.That transplanted sites at present commonly used has is cardiovascular, under peritoneal cavity and the kidney tunicle.With micro-capsule be expelled to cardiovascular in, micro-capsule can directly contact with blood, so fast to material reacting condition speed in the blood.But the large volume micro-capsule can not be injected directly into cardiovascular in.Though peritoneal cavity can be transplanted a large amount of micro-capsules, but because micro-capsule does not directly contact with blood in peritoneal cavity, therefore blunt to material reacting condition in the blood, and, make micro-capsule inner cell excretory material not to be diffused into outside the micro-capsule fast because the volume of micro-capsule is big.Reacting condition speed is fast to external world for cell in the by contrast little micro-capsule.And because the cubic capacity of transplanted sites is limited, the more little cell quantity that can transplant of micro-capsule volume is just many more.The shape of micro-capsule is inhomogeneous, can cause organizing and micro-capsule between frictional force increase, thereby the generation that causes inflammation descends the histocompatibility of micro-capsule.
Summary of the invention
The present invention is an improved device that designs for the problem that solves the existing apparatus existence.This device can be produced the micro-capsule that shape is even, volume is littler.
Fig. 1 is the overall picture of apparatus of the present invention, and the composition of this device is described with reference to figure 1:
Syringe 4 is made by nonconducting insulating material.Syringe 4 links to each other with syringe pump 1 by push rod 2.The fltting speed of syringe pump is 0.1-100ml/h, can connect 20,50, the syringe of 100ml.Syringe pump 1 promotes piston 3 by push rod 2, the fluent material that contains cell that piston 3 advances in the syringe.Entry needle 5 is connected on the syringe, and the specification of entry needle is 18-22G.Liquid arrives the end of entry needle by entry needle 5, forms drop 7.Lead 6 one ends are connected on the entry needle 5, and the other end connects the positive pole with the high-voltage pulse electrostatic power unit, so entry needle has positive electricity.Drop 7 falls into one and fills metal salt solution (CaCl
2) container 8 in, the bottom of container 8 links to each other with lead 9, lead 9 is connected in the negative pole of high-voltage pulse electrostatic power unit.Syringe pump 1 by clip 12, container 8 by clip 11 connect with iron stand 13 on, iron stand bottom 10 play the whole device of balance.
Fig. 2 is the circuit of high-voltage pulse static droplet generator, and with reference to the circuit pulse producer 14 generation high-frequency pulse signals of figure 2 explanation high pressure static electricity surge generators, modulation pulse generator 23 produces low frequency pulse signals.Two kinds of pulse signals are sent to modulator 15.Amplifier 16 is accepted the pulse signal of 15 outputs through high pressure and power magnification output 10KV high pressure.Power amplifier is made up of high power transistor, driving circuit, high voltage pulse transformer and current discharge circuit, guarantees the amplifier reliable operation.High pressure producer 17 is accepted 16 the continuously adjustable high pressure of pulse signal output 0-100KV.High pressure is by 6 and 9 of lead 18 (positive pole), 19 (negative poles) difference interface chart 1.Power supply is provided with primary source 20 and control power supply 21 respectively.By adjusting, realize the continuous adjusting of high pressure producer 17 output 0-100KV voltages to primary source 20 output voltage values.Sparking protector 22 shields.The parameter of this high-voltage pulse electrostatic power unit is as follows:
Voltage: 0-100KV is adjustable continuously; Pulse-repetition: 10-100HZ; Pulse length: 1-6ms.
Fig. 3 is the HV generator pulse waveform, with reference to the waveform of figure 3 explanation electricimpulses
Operating voltage Vs:0-100KV; Basic voltage Vb:220V
AC represents recurrence interval a: 10-100MS, and promptly frequency is 10-100HZ.
AB represents in the one-period working pulse action time; 1-6ms.There is this moment electrical forces to produce.
BC represents basic voltage 4-99ms action time.This section period does not have electrical forces.
Utilize high-voltage pulse static droplet generator provided by the invention to make the drop of diameter 50-450 μ m, this drop falls into the solution that has opposite charges and forms solid glue pearl.The high-voltage pulse electrostatic power unit can produce the static of varying strength, frequency, pulse length.Voltage has determined the size of drop suffered electrical forces in electric field.The frequency decision unit time, interior what pulse actions were in drop.Pulse length determines the time that a pulse internal electric field power plays a role.When surpassing, drop will fall when certain size promptly surpasses its charge threshold level.
The pictorial diagram of Fig. 4 high-voltage pulse static droplet generator.
Use this device and make micro-capsule by the following method;
But at first will be mixed with viable cell or living tissue gel material solution packs in the syringe.But this gel material is the polysaccharide material.Saccharan commonly used is a sodium alginate.Sodium alginate is grouped into by two kinds of one-tenth, is respectively guluronic acid (G) and mannuronic acid (M).Because the biocompatibility of guluronic acid is better than mannuronic acid, so the present invention uses the high sodium alginate of guluronic acid content, viscosity 50-1400CPS.Open syringe pump and electrostatic power unit, formed drop on the needle point of syringe, drop falls in the divalent metal salts solution in the effect of electrostatic field force.Metal ion generation colloidization in sodium alginate and the metal-salt forms the glue pearl.Metal salt solution commonly used is CaCl
2Solution also can be used BaCl
2Solution.CaCl
2Concentration be 1.5-2.0%.
Then the glue pearl is put into a kind of polyamino acid solution, because polyamino acid has negative charge and sodium alginate has positive charge, so semi-permeable membranes with selective permeability of G, M and polyamino acid generation ionomer effect formation.The molecular weight of sodium alginate, the molecular weight of polyamino acid, and all can influence the thickness and the permeability of semi-permeable membranes the duration of contact of the two.General along with the rising of molecular weight, the prolongation of duration of contact, the permeability of film descends, and the thickness of film rises, and soundness also rises.Otherwise the permeability of film rises, and thickness descends, and soundness also descends.Polyamino acid commonly used is poly-lysine (PLL) and poly-ornithine.The molecular weight control of general poly-lysine is at 1-30KDa, the most handy 17KDa.Molecular weight can not pass through greater than the material of 80KDa.Because polylysine is positively charged, and the intravital protein belt negative charge of machine, the two is easily in conjunction with reacting.Therefore the PLL reaction that easily causes inflammation in vivo.Therefore need on the PLL film, cover the material that one deck has biocompatibility.What we selected for use is sodium alginate, but identical concentration with the sodium alginate that begins usefulness is low.
The glue pearl that next will have semi-permeable membranes is put into the sodium alginate soln of lower concentration.So just formed APA micro-capsule with good biocompatibility.
At last micro-capsule is put into metal chelator such as Ca ionic sequestrant, Trisodium Citrate, EDTA.Sequestrant and micro-capsule intermediary calcium one SODIUM ALGINATE GUM pearl react, and calcium ion combines with sequestrant, and sodium alginate reverts to liquid state.Someone thinks that the micro-capsule permeability of liquefaction is good, helps the transmission and the infiltration of material, helps the survival of cell.
Apparatus of the present invention can prepare the micro-capsule of diameter from 50 μ m-450 μ m as required, these micro-capsule volumes are little, form evenly, size is consistent, can be applicable to bag by viable cell, for example pancreas islet, liver cell, thyroid cell, sexual cell, stem cell etc.The micro-capsule that this device is made can directly be transplanted with the method for injection and not need to perform the operation.Micro-capsule has the survival that good permeability and biocompatibility help cell, helps wrapping the cell performance biological effect of quilt.
Description of drawings
The overall picture of Fig. 1 device
The circuit of Fig. 2 high-voltage pulse static droplet generator
The pulse waveform of Fig. 3 HV generator
The pictorial diagram of Fig. 4 high-voltage pulse static droplet generator
Embodiment
The glue pearl that embodiment 1 makes mean diameter 400 μ m
Sodium alginate soln with 2% (viscosity 250CPS) is put into the syringe of 20-100 milliliter.Syringe is put into syringe pump, and syringe is connected with a 22G syringe needle, and its diameter is 0.37 μ m, 17 ° of pillow gradients.In the positive pole connection and needle point of HV generator, negative pole is connected in and fills 2%CaCl
2The bottom of the container of solution.Liquid level and needle point apart from 20mm, voltage 10KV, frequency 100HZ, pulse length 3ms, syringe pump speed 2ml/s.Open syringe pump and high-voltage pulse electrostatic power unit.Drop forms on needle point, and splashes into CaCl
2Form the glue pearl in the solution.Collect the glue pearl after 10 minutes, observe diameter at the microscopically that has micrometer.The glue pearl is complete smooth.
The glue pearl that embodiment 2 makes mean diameter 200 μ m
Syringe needle internal diameter 0.24 μ m, 0 ° of the long 100mm pillow of pin gradient.Other condition is with embodiment 1.Can produce the glue pearl of mean diameter 212 ± 14 μ m.
With 4 * 10
3Individual diameter is suspended in 2% sodium alginate soln that 4 milliliters of viscosity are 250CPS less than the pancreas islet of 300 μ m.Use the various parameters among the embodiment 1, the drop of making splashes into 2%Cacl
2In the solution.After 10 minutes, inhale and remove Cacl
2Solution.The glue pearl is 7.2 physiological saline cleaning with PH, 60 eye mesh screens filter, put into the 0.05%PLL that molecular weight is 17KDa, cultivated at least 6 minutes, discard PLL solution, the physiological saline cleaning with PH is 7.0 discards solution, putting into 0.05% sodium alginate soln and soak 9min, is that 7.4 physiological saline cleans with PH then.The micro-capsule of producing with this method is smooth complete, mean diameter 400 μ m.
Claims (10)
1 one kinds of devices that prepare microencapsulated cell, be made up of injecting systems, static generation systems and subsystem three parts: injecting systems is made up of automatic syringe pump (1), syringe (4), entry needle (5); The static generation systems is made up of high-voltage pulse static droplet generator, lead (6,9); Subsystem is made up of iron stand (13), clip (11,12), insulating vessel (8).
Device described in 2 claims 1, it is characterized in that: employed syringe capacity is 20-100ml, and the entry needle specification is 18-22G, 0-17 ° of its obliquity, the fltting speed of syringe pump is 0.1-100ml/h, and the operating voltage of high-voltage pulse static droplet generator is 0-100kV.
3 one kinds of methods of utilizing the described device of claim 1 to prepare microencapsulated cell, comprise: the sodium alginate soln that (1) will be mixed with viable cell is packed in the syringe, open syringe pump and electrostatic power unit, under the effect of the fltting speed 0.1-100ml/h of syringe pump and static droplet generator operating voltage 0-100kV, make and form drop on the entry needle, drop falls in the divalent metal salts solution in the effect of electrostatic field force, metal ion generation colloidization in sodium alginate and the metal salt solution forms the glue pearl; (2) the glue pearl is put into a kind of polyamino acid solution, sodium alginate has positive charge because polyamino acid has negative charge, sodium alginate and the effect of polyamino acid generation ionomer, thus form semi-permeable membranes with selective permeability; (3) the glue pearl that will have a semi-permeable membranes is put into the sodium alginate soln of lower concentration, sodium alginate is combined with not full cross-linked polyamino acid, thereby form one deck marine alga sorrel again outside semi-permeable membranes, has so just formed micro-capsule; (4) micro-capsule is put into metal chelator, metal chelator and micro-capsule intermediary divalent-metal ion-Lalgine glue pearl reacts, and divalent-metal ion combines with sequestrant, and Lalgine reverts to liquid state, thereby prepares microencapsulated cell.
Microencapsulated cell preparation method described in 4 claims 3 is characterized in that: cell can be somatocyte or sexual cell.
Microencapsulated cell preparation method described in 5 claims 3 is characterized in that: the divalent metal salts solution can be BaCl
2Or CaCl
2
Microencapsulated cell preparation method described in 6 claims 5 is characterized in that: CaCl
2The concentration of salts solution is 1.5-2%.
Microencapsulated cell preparation method described in 7 claims 3 is characterized in that: polyamino acid can be poly-lysine or poly-ornithine.
Microencapsulated cell preparation method described in 8 claims 7 is characterized in that: the molecular weight of poly-lysine is 1-30kDa.
Microencapsulated cell preparation method described in 9 claims 7 or 8 is characterized in that: the molecular weight of poly-lysine is 17KDa.
Microencapsulated cell preparation method described in 10 claims 3 is characterized in that: metal chelator can be Trisodium Citrate or EDTA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03148551 CN1277920C (en) | 2003-07-03 | 2003-07-03 | Device for making microencapsulation cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03148551 CN1277920C (en) | 2003-07-03 | 2003-07-03 | Device for making microencapsulation cell |
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| CN1566339A CN1566339A (en) | 2005-01-19 |
| CN1277920C true CN1277920C (en) | 2006-10-04 |
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| CN 03148551 Expired - Fee Related CN1277920C (en) | 2003-07-03 | 2003-07-03 | Device for making microencapsulation cell |
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104328106B (en) * | 2014-08-25 | 2017-01-18 | 湖南中医药大学 | Microcapsule preparation apparatus and method |
| CN104263632B (en) * | 2014-09-15 | 2016-06-29 | 中国科学院南海海洋研究所 | A kind of microorganism embedding microsphere continuous preparation device and method |
| CN106995784A (en) * | 2017-03-15 | 2017-08-01 | 柳州市妇幼保健院 | The culture drop producing device of Embryo Culture ware |
| CN107118964A (en) * | 2017-05-10 | 2017-09-01 | 王学军 | A kind of automatic cytological dimensional culture microcapsule generating apparatus and method |
| CN108207931B (en) * | 2017-12-05 | 2021-11-30 | 大连敏慧精益科技有限公司 | Method for freezing and preserving trace sperms |
| CN112956473B (en) * | 2021-03-10 | 2023-05-26 | 中科强参(吉林)科技有限公司 | Artificial transparent belt and preparation method thereof |
| CN114317243B (en) * | 2021-12-31 | 2024-07-09 | 北京工业大学 | Method and apparatus for on-demand cell encapsulation by coaxial electrospray of air pressure pulse liquid supply |
| CN114921343B (en) * | 2022-06-28 | 2023-09-05 | 中国科学院苏州生物医学工程技术研究所 | Cell gel microsphere generating device based on high-voltage pulse electric field |
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