CN1566339A - Device for making microencapsulation cell - Google Patents
Device for making microencapsulation cell Download PDFInfo
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- CN1566339A CN1566339A CN 03148551 CN03148551A CN1566339A CN 1566339 A CN1566339 A CN 1566339A CN 03148551 CN03148551 CN 03148551 CN 03148551 A CN03148551 A CN 03148551A CN 1566339 A CN1566339 A CN 1566339A
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- cell
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- sodium alginate
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Abstract
The invention discloses a device for making microencapsulation cell, which comprises an injection system, a high-voltage pulse generator and an accessory system. The microcapsules prepared thereby can be applied for cell culturing and cell transplantation.
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 coated cell, carries out cell cultures and Transplanted cells as islet cells, liver cell etc.
Background technology
Micro-capsule has selective permeability, and 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 rapidly to outside the micro-capsule because the volume of micro-capsule is big.By contrast, reacting condition speed is fast to external world for the cell in the 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 also 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 is designing 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, and piston 3 advances the fluent material that contains cell in the syringe.Entry needle 5 is connected on the syringe, and the specification of entry needle is 18-22G.Liquid forms drop 7 by the end of entry needle 5 arrival entry needles.Lead 6 one ends are connected on the entry needle 5, and the other end is connected in the positive pole of 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 is connected on the iron stand 13 by clip 7 by clip 12, container 8, and the whole device of balance is played in the bottom 10 of iron stand.
Fig. 2 is the circuit of high-voltage pulse static droplet generator, and the circuit of high pressure static electricity surge generators is described with reference to figure 2:
Surge generator 14 produces high-frequency pulse signal, and modulation pulse generator 23 produces low frequency pulse signal.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 pulse waveform of HV generator, and the waveform of electricimpulses is described with reference to figure 3:
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 can 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.
Fig. 4 is the pictorial diagram of high-voltage pulse static droplet generator.
Use this device and make micro-capsule by the following method:
But the gel material solution that at first will be mixed with viable cell is packed 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, form 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 commonly used is CaCl
2, also can use BaCl
2CaCl
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-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 to 450 μ m as required, and these micro-capsule volumes are little, shape even, 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 good permeability and biocompatibility, helps the survival of cell, helps wrapping the cell performance biological effect of quilt.
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, its diameter 0.37 μ m, 17 ° of pillow gradients.The positive pole of HV generator is connected on the needle point, and 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.
Embodiment 3 makes the micro-capsule that contains pancreas islet
With 4 * 10
3Individual diameter is suspended in 2% sodium alginate soln of 4 milliliters of viscosity 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.Inhale after 10 minutes and remove CaCl
2Solution.The glue pearl is cleaned with the physiological saline of PH 7.2,60 eye mesh screens filter, put into the 0.05%PLL of molecular weight 17KDa, cultivated at least 6 minutes, discard PLL solution, the physiological saline cleaning with PH 7.0 discards solution, put into 0.05% sodium alginate soln and soaked 9 minutes, the physiological saline with PH 7.4 cleans then.The micro-capsule of producing with this method is smooth complete, mean diameter 400 μ m.
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
Claims (10)
1 the present invention is a kind of device of making microencapsulated cell.This device is a high-voltage pulse static droplet generator, is made up of injecting systems, static generation systems, subsystem three parts.It is characterized in that to prepare the micro-capsule of 50-1000 micron.
Said injecting systems in 2 claims 1 is characterized in that being made up of automatic syringe pump, syringe, syringe needle.
Said static generation systems in 3 claims 1 is characterized in that being made up of high-voltage pulse electrostatic power unit, lead.
Said subsystem is made up of iron stand, clip, insulating vessel in 4 claims 1.
Said drop in 5 claims 1, but it is characterized in that forming by the material of gel.But the gel material can be the polysaccharide material.The polysaccharide material can be a sodium alginate.
After said sodium alginate forms a certain size drop in 6 claims 5, with a kind of salts solution reaction that contains divalent-metal ion colloidization taking place, forms the glue pearl.Said divalent-metal ion can be Ca
2+And Ba
2+Salts solution can be CaCl
2And BaCl
2Solution.
Glue pearl that forms in 7 claims 6 and the reaction of a kind of polyamino acid generation ionomer of oppositely charged form semi-permeable membranes on the surface.Said polyamino acid is a poly-lysine.
The glue pearl and the good saccharan material generation ionic reaction of biocompatibility that have semi-permeable membranes in 8 claims 7, purpose are to cover poly-lysine.
Said saccharan material in 9 claims 8 is characterized in that it can being sodium alginate.
Said cell in 10 claims 1 is characterized in that it can being somatocyte, also can be sexual cell.
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 |
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| 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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| Publication Number | Publication Date |
|---|---|
| CN1566339A true CN1566339A (en) | 2005-01-19 |
| CN1277920C CN1277920C (en) | 2006-10-04 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 03148551 Expired - Fee Related CN1277920C (en) | 2003-07-03 | 2003-07-03 | Device for making microencapsulation cell |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104263632A (en) * | 2014-09-15 | 2015-01-07 | 中国科学院南海海洋研究所 | Microbe embedded microsphere continuous preparation device and method |
| CN104328106A (en) * | 2014-08-25 | 2015-02-04 | 湖南中医药大学 | Microcapsule preparation apparatus 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 |
| CN108207931A (en) * | 2017-12-05 | 2018-06-29 | 中国科学院大连化学物理研究所 | A kind of micro sperm cryopreservation method |
| CN112956473A (en) * | 2021-03-10 | 2021-06-15 | 吉林省强参生物技术有限公司 | Artificial transparent belt and preparation method thereof |
| CN114317243A (en) * | 2021-12-31 | 2022-04-12 | 北京工业大学 | Coaxial electrospray on-demand cell encapsulation method and device for pneumatic pulse liquid supply |
| CN114921343A (en) * | 2022-06-28 | 2022-08-19 | 中国科学院苏州生物医学工程技术研究所 | Cell gel microsphere generation device based on high-voltage pulse electric field |
-
2003
- 2003-07-03 CN CN 03148551 patent/CN1277920C/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104328106A (en) * | 2014-08-25 | 2015-02-04 | 湖南中医药大学 | Microcapsule preparation apparatus and method |
| CN104263632A (en) * | 2014-09-15 | 2015-01-07 | 中国科学院南海海洋研究所 | Microbe embedded 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 |
| CN108207931A (en) * | 2017-12-05 | 2018-06-29 | 中国科学院大连化学物理研究所 | A kind of micro sperm cryopreservation 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 |
| CN112956473A (en) * | 2021-03-10 | 2021-06-15 | 吉林省强参生物技术有限公司 | Artificial transparent belt and preparation method thereof |
| CN114317243A (en) * | 2021-12-31 | 2022-04-12 | 北京工业大学 | Coaxial electrospray on-demand cell encapsulation method and device for pneumatic pulse liquid supply |
| CN114921343A (en) * | 2022-06-28 | 2022-08-19 | 中国科学院苏州生物医学工程技术研究所 | Cell gel microsphere generation device based on high-voltage pulse electric field |
| 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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|---|---|
| CN1277920C (en) | 2006-10-04 |
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Granted publication date: 20061004 Termination date: 20170703 |