CN1438313A - Method for preparing microcapsulized functional cell - Google Patents
Method for preparing microcapsulized functional cell Download PDFInfo
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- CN1438313A CN1438313A CN 03115715 CN03115715A CN1438313A CN 1438313 A CN1438313 A CN 1438313A CN 03115715 CN03115715 CN 03115715 CN 03115715 A CN03115715 A CN 03115715A CN 1438313 A CN1438313 A CN 1438313A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
- C12M33/06—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles for multiple inoculation or multiple collection of samples
Abstract
The invention is a method of making microencapsulated functional cell. It mixes evenly the filtered and axenic bubble enveloping liquid and good cells in proportion, then places the mixture in the multichannel generating equipment, spray the cell suspended liquid to form bubble, and then processes and reserves. The generating equipment includes injection pump, connecting hose (2-1), union joint, hose core, gland, sealing ring, fixing ring, jacket and compressed gas generator.
Description
Technical field
The invention belongs to the biotherapy technical field, relate to a kind of biological micro-capsule technology of preparing, relate in particular to a kind of preparation method of microencapsulation functioning cell, can prepare the microencapsulation functioning cell of height histocompatibility, permeability and stability, can effectively be applied to the biotherapy of tumour.
Technical background
In recent years, the 4th kind of biotherapy for the treatment of pattern further developed as malignant tumour, and it is important that the status in oncotherapy more and more seems.The wherein application of the genetically engineered cell factor, immunotherapy of tumors and therapy of tumor are studied and are most widely used, and have obtained certain effect.But,, guarantee effectively to treat concentration and must continue heavy dose of intravenously administrable or be interrupted subcutaneous injection because cytokine is shorter biological half-life in vivo.And heavy dose of medication often can cause serious toxic side effect; The purity and the activity of recombinant cytokine also are difficult for satisfactory simultaneously.For this reason, use the microencapsulated cell technology and combine with anti-tumor activity molecular gene engineering under immune isolation, discussion is the antitumor action of the microencapsulation genetically engineered cell of secrete cytokines for a long time.
Microencapsulated cell (microencapsulated cells) is that viable cell is wrapped in sacculus in the semi-permeable membranes.Implanting to human body behind the functioning cell microencapsulated encapsulate can be kept survival, and keep normal physiological function, thereby acquisition can be secreted the microencapsulation genetically modified cell of purpose activated protein in vivo for a long time, constantly, reach the purpose of treatment disease.Whether successful use one of microencapsulation functioning cell treatment disease key is to prepare the micro-capsule with good organization's consistency, permeability and stability.
The micro-capsule parcel is highly effective immune isolation technology, can be applicable to the many Transplanted cellss that can secrete biologically active substance and bring into play function, can also wrap up micromolecular enzyme and other active substance performance immanoprotection action.The microencapsulated cell technology can solve external transgenic cell and is transplanted to problems such as the back survival rate is low in the body, the cytokine transformation period is short with engineered the combination, can be biotherapy an effective new way is provided.
At present, microencapsulation technology is mainly used in: domestic microencapsulation islet cell transplantation treatment diabetes and the microencapsulation of pheochromocyte is used for the research etc. of Parkinson's disease and pain; The external research that also has application microencapsulation genetically modified cell to be used for disease treatment, target gene mainly contains human growth hormone (hGH) gene, growth factor of human nerve (hNGF) gene etc.According to the literature, the acquisition of micro-capsule at present is to reach " two-chamber generator for microcapsules " preparation by " micro-capsule electrostatic power unit ".
Summary of the invention
An object of the present invention is to provide a kind of preparation method of microencapsulation functioning cell, can prepare microencapsulation functioning cell, be effectively applied to tumor biotherapy with height histocompatibility, permeability and stability.
Preparation method of the present invention comprises: the micro-capsule parcel liquid and the growth conditions good cell of getting filtration sterilization, with functioning cell on request mixed in micro-capsule parcel liquid, mixing in the mixed solution inhalation syringe, places on the micro-injection pump, connection is also opened hyperchannel micro-capsule generating unit, cell suspending liquid is sprayed the formation micro-capsule, and fall into receiving tank, collect micro-capsule, handle, standby.
Used micro-capsule parcel liquid can be sodium alginate, poly-lysine, agarose, polypropylene amine etc.
Can prepare the micro-capsule (φ 0.35-0.75mm) of different diameter size.
Another object of the present invention provides a kind of hyperchannel micro-capsule generating unit that is used to prepare above-mentioned microencapsulation functioning cell.
This hyperchannel micro-capsule generating unit comprises: the piston-type syringe pump, pipe connecting, tube stub, tube core, gland, sealing-ring, fixed ring, overcoat, compressed gas generator, a pipeline of pipe connecting is connected with the piston-type syringe pump, all the other each pipelines connect the tube core of respective counts by tube stub, tube core passes gland successively, sealing-ring, the center of fixed ring, sealing-ring is between gland and fixed ring, overcoat is placed in tube core, sealing-ring, outside the fixed ring, a path of another pipe connecting is connected with compressed gas generator, and all the other each pipelines communicate with space between overcoat and tube core by tube stub.
Pipe connecting has many pipelines, forms many paths.
Between overcoat and tube core the space is arranged.
The present invention has the following advantages: (1) micro-capsule making processes is simple relatively, and preparation speed is fast, can be used for a large amount of micro-capsule of preparation fast; (2) Zhi Bei micro-capsule is little to the influence of parcel cell activity; (3) can prepare the micro-capsule (φ 0.35-0.75mm) of different diameter size by adjusting relevant parameter; (4) uniformity of micro-capsule, stability, permeability and good biocompatibility; (5) above these characteristics can effectively provide the microencapsulation that is applied to biotherapy functioning cell.(6) hyperchannel micro-capsule generating unit reasonable in design, novelty; (7) the device preparation is easy, and cost is low.
Description of drawings
Fig. 1 is a kind of structural representation of hyperchannel micro-capsule generating unit of the present invention.
Fig. 2 is microencapsulation CHO-mIFN-gamma cells figure.
Fig. 3 is CHO/mIFN-gamma cells and cell microcapsule scatter chart thereof.
Embodiment
Below with reference to specific embodiments and the drawings, the present invention is further illustrated.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.
Embodiment 1
Referring to Fig. 1, a kind of structure of hyperchannel micro-capsule generating unit comprises: piston-type syringe pump 1, pipe connecting 2 and 2-1, tube stub 3, tube core 4, gland 5, sealing-ring 6, fixed ring 7, overcoat 8, compressed gas generator 9, pipe connecting 2 has a plurality of paths, one of them path is connected with piston-type syringe pump 1, all the other each paths connect the tube core 4 of respective counts by tube stub 3, the other end of tube core 4 is the outlet of micro-capsule, tube core 4 passes gland 5 successively, sealing-ring 6, the center of fixed ring 7, sealing-ring 6 is between gland 5 and fixed ring 7, overcoat 8 is placed in tube core 4, sealing-ring 6, outside the fixed ring 7, form right cylinder, the right cylinder upper end covers with gland 5, the lower end becomes the outlet of micro-capsule, 4 of overcoat 8 and tube cores have the space, and pipe connecting 2-1 has a plurality of paths, and one of them path is connected with compressed gas generator 9, all the other each paths pass overcoat 8 by tube stub 3, communicate with the space of 4 of overcoat 8 and tube cores.
The microencapsulated encapsulate of cell: get growth conditions good, reach 90% Chinese hamster ovary celI that merges, with 0.25% tryptic digestion physiological saline washing 2 times, centrifugal, counting, the micro-capsule of getting filtration sterilization wraps up the liquid sodium alginate, with cell in proportion (1.5 * 10
9L
-1Sodium alginate) is mixed in the sodium alginate soln, blows and beats mixing gently.With hyperchannel micro-capsule generating unit cell suspending liquid is spurted into 4.9% BaCl is housed
2In the Receiving bin of solution (pH7.4), behind the 10min, with 1 * PBS liquid washing 3 times.Collect micro-capsule, dynamic observe the existence situation of capsule inner cell under the mirror.Place the DMEM that contains 10% calf serum, cultivate (37 ℃ of 5% CO in the incubator
2) standby.
Used micro-capsule parcel liquid can also be poly-lysine, agarose, polypropylene amine etc.
The airshed of the injection rate that can be by regulating piston formula syringe pump and the pressurized gas of compressed gas generator is prepared the micro-capsule (φ 0.35-0.75mm) of different diameter size.
Mensuration (one) experiment material of embodiment 3 sodium alginate micro-capsule physicochemical properties
Instrument material: laser scanning co-focusing microscope (LSCM 510); Constant temperature oscillator TH2-c; The dextran of isosulfocyanic acid fluorescence mark (FITC-dextran, FD) (FD-40, FD-75 and FD-167).The goat anti-mouse igg of FITC mark (F-IgG), trypsinase; The DMEM substratum, new-born calf serum.Chinese hamster ovary celI.
Laboratory animal: BALB/c mouse, age in 8-10 week, body weight 20-25g.(2) permeability of measuring method 1. micro-capsules of micro-capsule physicochemical property is measured static the observation
The various micro-capsules of above-mentioned preparation are suspended in 0.5ml respectively to be contained in FD-40, FD-75 or FD-167 solution (being 100mg/L) and the F-IgG solution (100mg/L), room temperature, lucifuge are placed 24hr, and constantly shake, fluorescein-labelled thing can fully be diffused in the micro-capsule.Adjust laser scanning co-focusing microscope (LSCM 510), and preheating 15min.A little is above-mentionedly placed on the disposable culture dish of 35mm as for the micro-capsule in the liquid, under LSCM 510 ordinary light, focus on micro-capsule, and then be replaced with LASER Light Source, on display screen, observe the laser co-focusing image, and preserve the picture concerned data.Dynamic observe
To newly prepare micro-capsule to be measured and place on the disposable culture dish of 35mm, be added dropwise to an amount of FD-40, FD-75 or FD-167 solution (being 100mg/L) respectively successively.LSCM is set to the time scan mode immediately, scans an images every 1min, continuous sweep 30min.Dynamic observe the situation of FD, and preserve the picture concerned data to micro-capsule internal diffusion to be measured.The permeability of cell microcapsule is observed
Preparation includes the micro-capsule of cell, place DMEM substratum (containing 10% new-born calf serum) hatch 24hr (37 ℃, 5%CO
2).With the empty micro-capsule through same processing is contrast.Carry out the permeability mensuration of micro-capsule then as stated above with LSCM.2. the mensuration of micro-capsule stability
Inject speed by above-mentioned flow process by adjusting oxygen flow and micro-injection pump, prepare little micro-capsule (diameter 0.40-0.50mm) and big micro-capsule (0.70-0.80mm).Micro-capsule with 1 * PBS flushing 3 times, is suspended again with physiological saline.Get 12 aseptic centrifuge tubes of 10ml, 6 pipes suck the above-mentioned suspension that 3ml contains little micro-capsule about 1000 approximately, and 6 pipes suck the big microcapsule suspension of same amount in addition.Place constant temperature oscillator to shake in 37 ℃ of 150 times/min, the concussion time is respectively 8,16,24,32,40 and 48hr.Under fiberscope, observe the micro-capsule form then and calculate breakage rate.3. the mensuration of micro-capsule biocompatibility
Get 8 BALB/C mice, subcutaneous injection NIH3T3 cell microcapsule, about 1000/every.Respectively at 2,4,8 and 12 weeks respectively getting 2 mouse, put to death the careful skin that cuts in back, take out subcutaneous micro-capsule at former injection position.Use normal saline flushing, examine form, understand micro-capsule in the survival condition that is subjected to intravital stability and capsule inner cell at microscopically.Promptly observe the biocompatibility of micro-capsule.(3) the static observations of the permeability of the permeability measurement result 1.1 empty micro-capsules of result's 1. micro-capsules of micro-capsule physicochemical property mensuration
The empty micro-capsule that the sodium alginate of employing 1.5% and 2% is made, the FD or the F-IgG of itself and different molecular weight are hatched 24hr altogether, the image that records with laser confocal microscope shows: the burnt visual micro-capsule clear-cut of the copolymerization of FD-75, FD-167 and F-IgG, no obvious fluorescence bright spot in the capsule shows that molecular-weight average is that 75kD above dextran and immunoglobulin IgG can not diffuse in the capsule by microcapsule membrane.The inside and outside basically identical of fluorescence intensity micro-capsule of FD-40 shows that molecular weight is that the dextran of 40kD can freely enter micro-capsule.1.2 FD-40 dynamic observes to the micro-capsule internal diffusion
Utilize the time scan function of LSCM can dynamic observe the diffusion process of FD-40 in micro-capsule.1.3 the permeability of cell microcapsule
The Chinese hamster ovary celI micro-capsule hatched 24hr in containing the DMEM substratum of 10% new-born calf serum after, with LSCM it is carried out permeability again and measure.The result shows the permeability no significant difference of Chinese hamster ovary celI micro-capsule and empty micro-capsule.2. the measurement result of micro-capsule stability
This experimental result shows: the micro-capsule diameter is relevant with its stability, and the big micro-capsule (diameter 0.75mm) of little micro-capsule (diameter 0.45mm) has better stability (table 1).
The dissimilar micro-capsules of table 1 are at the breakage rate (%) of different time
Time (hr)
Biocompatibility measurement result 3.1 micro-capsules of 8 16 24 32 40 48 big micro-capsule (diameter 0.75mm) 3.8 4.9 6.5 8.6 11.2 15.3 little micro-capsules (diameter 0.45mm), 1.1 1.65 1.8 2.1 3.2 5.63. micro-capsules are implanted into subcutaneous back 2 weeks, 4 weeks of mouse, 8 weeks took out micro-capsule respectively, under light microscopic, observe complete form, rounded, cyst wall is bright and clean, does not see that fibrosis holds.Micro-capsule inner cell still survive well (take out the back in the micro-capsule and continue to cultivate in conventional DMEM substratum, cell still can be bred).3.2 micro-capsule is implanted into the subcutaneous back of mouse during 12 weeks, most micro-capsule forms are still complete, rounded, and cyst wall is bright and clean, do not see that fibrosis holds; The small part micro-capsule has distortion, and cyst wall thickens.The micro-capsule that reclaims placed in the conventional DMEM substratum cultivate, the micro-capsule inner cell is still survived well.
Antitumor research (one) material of embodiment 4 microencapsulation mIFN-γ genetically modified cells
Sodium alginate (Alginate Sodium), bariumchloride (BaCl
2), the micro-strainer in various apertures, mIFN-γ ELISA detection kit, hyperchannel micro-capsule generating unit device provided by the invention, full-automatic microplate reader, BALB/C mice, male, 6-8wk, body weight 20-25g, CHO-mIFN-gamma cells.(2) preparation of microencapsulation CHO/IFN-gamma cells and IFN-γ thereof discharge and measure
Sodium alginate is disposed 1.5% solution, respectively with the micro-strainer of 1.2,0.8,0.45 and 0.22 μ m filter, degerming.CHO/mIFN-γ genetically modified cell is mixed in proportion in sodium alginate soln, and preparing diameter with hyperchannel micro-capsule generating unit device provided by the invention with reference to the method for embodiment 2 is that microencapsulated cell about 0.45mm is in 4.9% BaCl
2In the solution.1 * PBS gives a baby a bath on the third day after its birth inferior, places the RPMI1640 that contains 10% calf serum, 37 ℃ of 5%CO
2Cultivate in the incubator.Dynamic observe the existence situation of capsule inner cell under the mirror.Collected culture supernatant every 2 hours ,-20C preserves, and with the burst size of ELISA method detection IFN-γ, draws release profiles.(3) mice with tumor model and treatment thereof
After washing twice with RPMI-1640, the CT26 cell that will be in logarithmic phase is suspended among the serum-free RPMI-1640 (2 * 10
6/ ml), and in mouse right rear leg subcutaneous injection 0.1ml, promptly 2 * 10
5Individual CT26 cell.80 tumor-bearing mices are divided into 4 groups at random in second day, 20 every group, give different treatments respectively.Each 10 mouse of getting at random behind treatment 15d in each group are put to death, and pluck the variation that the eyeball bloodletting detects its immunologic function before the execution.All the other mouse are observed long-term surviving.
1. RPMI-1640 organizes: subcutaneous injection RPMI-1640;
2. Chinese hamster ovary celI micro-capsule group: subcutaneous injection CHO micro-capsule, 1 * 10
5Individual cell/only;
3. CHO/LacZ/Ad micro-capsule group: subcutaneous injection CHO/Ad/lacZ cell microcapsule, 1 * 10
5Individual cell/only;
4. CHO/IFN-γ micro-capsule group; Subcutaneous injection CHO/IFN-gamma cells micro-capsule, 1 * 10
5Individual cell/only.(4) result
1. the preparation of microencapsulation CHO/mIFN-gamma cells
Adopt hyperchannel micro-capsule generating unit provided by the invention,, prepared the micro-capsule (referring to Fig. 2) of big or small homogeneous, the about 0.5mm of diameter, show that the micro-capsule inner cell is evenly distributed with reference to the method for embodiment 2.
2. IFN-γ level in microencapsulated cell and the CHO/mIFN-gamma cells culture supernatant
It is micro-capsule about 0.5mm that CHO/mIFN-γ is rolled into diameter, detected mIFN-γ in the vitro culture supernatant of CHO/mIFN-gamma cells and microencapsulation CHO/mIFN-gamma cells, reach the peak after 20 hours in its transfection and be respectively 81.2 and 78.9ng/ml, draw out secretion curve (referring to Fig. 3).There is not significant difference (P＜0.05) between two groups.Express continually and steadily subsequently, 14d after the transfection still can detect mIFN-γ in the culture supernatant.And transfection empty carrier (CH0/LacZ) and Chinese hamster ovary celI do not detect IFN-γ.Show adenovirus with IFN-γ gene transfection to Chinese hamster ovary celI and be effective expression.But the survival of capsule inner cell long period also continues to produce mIFN-γ, and mIFN-γ can free penetrating sodium alginate micro-capsule film.
3. microencapsulation CHO/IFN-gamma cells is treated back mice with tumor serum cytokines level
Tumor-bearing mice is 2 weeks after the treatment of microencapsulation CHO-IFN-gamma cells, and treatment group serum mIFN-γ is high level expression, compare with control group, RPMI-1640 group and lacZ group, and difference all has significance (P＜0.05).(table 2)
Table 2 microencapsulated cell treatment mice with tumor serum I FN-γ level (x ± s) (pg/ml) after 15 days
The little CHO/LacZ/A CHO/IFN-of RPMI-1640 Chinese hamster ovary celI γ capsule
Capsule d capsule
205.1±63.9 197.6 ± 239.2 ± 2673.8 ±
80.3 71.1 576.2*
Compare with control group, RPMI-1640 group and lacZ group, n=20,
*P＜0.01 the present invention is described in conjunction with most preferred embodiment, yet after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims institute restricted portion equally.
The partial reference document 1.Leblond FA that the present invention relates to, Tessier J, Halle JP.Quantitative method for the evaluation of biomicrocapsule resistance to mechanical stress.Biomaterials, 1996,17 (21): 2097-2102.2.Peirone M, Ross CJ, Hortelano G et al.Encapsulation of various recombinant mammalian cell types in different alginate microcapsules.J Biomed Mater Res.1998,42 (4): 587-596.3.Hobbs HA, Kendall WF, Darrabie M, et al.Prevention of morphological changes in alginate microcapsules for islet xenotransplantation.JInvestig Med, 2001,49 (6): 572-575.4.Charles K, Harland RC, Ching D, et al.torage and microencapsulation of islets for transplantation.Cell-Transplant.2000,9 (1): 33-38.5.Uludag H, De-Vos P, Tresco PA.Technology of mammalian cell encapsulation.Adv Drug Deliv Rev.2000,42 (1-2): 29-64.6.Rayat GR, Rajotte RV, Ao Z, et al.Microencapsulation of neonatal porcine islets:Protection from human antibody/complenent-mediated cytolysis in vitro and long-term reversal of diabetes in nude mice.Transplantation, 2000,69:1084-1890.7.Tai IT, Sun AM.Microencapsulation of recombinant cells:A new delivery system for gene therap y.FASEB, 1993,7:1061-1069.8.Uludag H, De Vos P, Patrick A, et al.Technology of mammalian cell encapsulation. Advanced Drug Delivery Revies, 2000,42,29-64.9.Aebischer P, Lysaght MJ.Immunoisolation and cellular xenotransplantation.Xeno, 1995,3:43-46.10.Ezzell C.Tissue enginnering and the human body shop:encapsulated-cell transplants enter the clinic.J NIH Research.1995; 7:47-50.11.Chang PL, Shen N, Westcott AJ.Delivery of recombinant gene products with microencapsulated cells in vivo.Hum Gene Ther.1993,4 (4): 433-440.12.Calafiore R.Transplantation of microencapsulated pancreatic human islets for therapy of diabetes meilitus.ASAIO, 1992; 38:34.13.Cai ZH, Shi ZQ, Sherman M, et al.Development and evaluation of a system of microcapsulation of primary rat hepatocytes.Hepatology, 1989; 10:855.14.Tresco PA, Winn SR, Aebischer P.Polymer encapsulated neurotransmitter secreting cell. Potiential treatment for Pakinson ' s disease.ASAIO J, 1992; 38:17.15.Hoffman D, Brerakefield XO, Short MP, et al.Transplantation of polymer-encapsulated cell line genetically engineered to release.NGF Exp Neurol, 1993; 122:100.16.Pheips CH, Gage FH, Growdon JH, et al.Potential use of nerve growth factor to treat Alzheimer ' s disease.Neurobiol ofAging, 1989; 10:205-207.17.Joki T, Machluf M, Atala A, et al.Continuous release of endostatin from microencapsulated engineered cells for tumor therapy.Nat Biotechnol.2001,19 (1): 35-39.18.Read TA, Sorensen DR, Mahesparan R, et al.Local endostatin treatment of gliomas administered by microencapsulated producer cells.Nat Biotechnol, 2001,19 (1): 29-34.19.De Vos P, De Haan BJ, Wolters GHJ, et al.Improved biocompatibility but limited graft survival after purification of alginate for microencapsulation of pancreatic islets.Diabetologia, 1997; 40:262-270.20.Kulseng B, Skjak BG, Ryan L, et al.Transplantation of alginate microcapsules:Generation of antibodies against alginates and encapsulated porcine islet-like cell clusters.Translantation, 1999,67:978-984.21. in wise, cold uncommon sage, Wei Yuhua etc.Alginate purity is to the influence of hepatocyte function in rat micro-capsule fibrosis and the capsule. Chinese Medical Journal, 1999, (6): 460-462.22. cold uncommon sage, Wei Yuhua, Liu Ji is superfine.Alginate purity is to the Fibrotic influence of micro-capsule in the microencapsulated hepatocyte intraperitoneal transplantation. Chinese surgical magazine, 2000,38 (4): 300-303.23.Zhang WJ, Marx SK, Lane C.HOH077 reduces fibrotic overgrowth around the barium alginate microcapsules.Transplant Proc, 2000,32:206-209.24.Fritschy WM, De Vos P, Groen H, et al.The capsular overgrowth on microencapsulated pancreatic islet grafts in streptozotocin and autoimmune diabetic rats.Transplant Int.1994; 7:264-271.25.Xue Y, Gao J, Wang Z, et al.Microencapsulated bovine chromaffin cell xenografts into hemiparkinsonian rats:a drug-induced rotational behavior and histological changes analysis. Artif Organs.2001,25 (2): 131-135.26.Tabler C, Wilks K, Sambanis A, et al.The effects of alginate composition on encapsulated betaTC3 cells.Biomaterials.2001,22 (11): 1301-1310.
Claims (7)
1. the preparation method of microencapsulation functioning cell, it is characterized in that: get the micro-capsule parcel liquid and the growth conditions good cell of filtration sterilization, with functioning cell on request mixed in micro-capsule parcel liquid, mixing, in the mixed solution inhalation syringe, place on the micro-injection pump, connect and open hyperchannel micro-capsule generating unit, cell suspending liquid is sprayed form micro-capsule, and fall into receiving tank, collect micro-capsule, handle, standby.
2. the preparation method of microencapsulation functioning cell according to claim 1 is characterized in that: used micro-capsule parcel liquid can be sodium alginate, poly-lysine, agarose, polypropylene amine etc.
3. the preparation method of microencapsulation functioning cell according to claim 1 is characterized in that: the micro-capsule (φ 0.35-0.75mm) that can prepare the different diameter size.
4. hyperchannel micro-capsule generating unit, comprise: piston-type syringe pump (1), compressed gas generator (9), it is characterized in that: also comprise pipe connecting (2) (2-1), tube stub (3), tube core (4), gland (5), sealing-ring (6), fixed ring (7), overcoat (8), a pipeline of pipe connecting (2) is connected with piston-type syringe pump (1), all the other each pipelines connect corresponding tube core (4) by tube stub (3), tube core (4) passes gland (5) successively, sealing-ring (6), the center of fixed ring (7), sealing-ring (6) is positioned between gland (5) and the fixed ring (7), overcoat (8) is placed in tube core (4), sealing-ring (5), outside the fixed ring (6), a pipeline of pipe connecting (2-1) is connected with compressed gas generator (9), and all the other each pipelines are connected with overcoat (8) by tube stub (3).
5. hyperchannel micro-capsule generating unit according to claim 4 is characterized in that: pipe connecting (2) (2-1) has a plurality of pipelines, forms many paths.
6. hyperchannel micro-capsule generating unit according to claim 4 is characterized in that: between overcoat (8) and tube core (4) space is arranged.
7. according to claim 4 and 6 described hyperchannel micro-capsule generating units, it is characterized in that: all the other each pipelines of pipe connecting (2-1) pass overcoat (8), communicate with space between overcoat (8) and tube core (4).
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| CN106237327A (en) * | 2015-06-12 | 2016-12-21 | 林梓栋 | A kind of broad-spectrum tumor vaccine adjuvant and preparation method thereof and preparation facilities |
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