CN108998378B - Integrated primary cell digestion and separation bottle - Google Patents
Integrated primary cell digestion and separation bottle Download PDFInfo
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- CN108998378B CN108998378B CN201811146980.0A CN201811146980A CN108998378B CN 108998378 B CN108998378 B CN 108998378B CN 201811146980 A CN201811146980 A CN 201811146980A CN 108998378 B CN108998378 B CN 108998378B
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- 230000029087 digestion Effects 0.000 title claims abstract description 100
- 238000000926 separation method Methods 0.000 title claims abstract description 22
- 230000001681 protective effect Effects 0.000 claims abstract description 16
- 230000001079 digestive effect Effects 0.000 claims description 11
- 238000000605 extraction Methods 0.000 abstract description 10
- 238000001914 filtration Methods 0.000 abstract description 3
- 238000011085 pressure filtration Methods 0.000 abstract description 3
- 238000004062 sedimentation Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 28
- 102000038379 digestive enzymes Human genes 0.000 description 6
- 108091007734 digestive enzymes Proteins 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000010586 diagram Methods 0.000 description 4
- 210000003739 neck Anatomy 0.000 description 4
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 229960002897 heparin Drugs 0.000 description 3
- 229920000669 heparin Polymers 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000005054 agglomeration Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 210000002445 nipple Anatomy 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
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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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/08—Flask, bottle or test tube
-
- 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
- C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Sustainable Development (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Clinical Laboratory Science (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses an integrated primary cell digestion and separation bottle, which is characterized in that: including switch formula filter tip, aseptic protective sheath, malleation formula digestion bottle and digestion bottle support, the cover is equipped with can be dismantled to switch formula filter tip one end aseptic protective sheath, the other end with the connection can be dismantled to malleation formula digestion bottle, and be equipped with first filter screen, side at switch formula filter tip's inside and be equipped with rotary type check valve, be equipped with application of sample mouth and malleation mouth on the bottle of malleation formula digestion bottle, and be equipped with the second filter screen at the one end that this malleation formula digestion bottle and switch formula filter tip are connected. The invention provides an integrated digestion and filtration process, which effectively shortens a learning curve, shortens operation time, improves insufficient cell extraction caused by primary cell sedimentation with overlong operation time, and ensures convenience and high efficiency of primary cell extraction through a unique one-way switch control and positive pressure filtration mode.
Description
Technical Field
The invention relates to a cell digestion bottle, in particular to an integrated primary cell digestion separation bottle.
Background
Primary cells are cells isolated and cultured immediately after removal from an animal or human body, and generally, the 1 st generation cells and cells within 10 passages are called primary cell culture. Primary cells are widely used in various fields of molecular, cell biology and biomedical research, and have an irreplaceable role in the biomedical field because they retain the nature of cells of the body itself. Current primary cell extraction includes digestion and separation methods, tissue attachment methods, and the like. Unfortunately, currently, there is no universal efficient digestion separation vessel for digestion separation processes, and autoclaved glass vessels are commonly used as digestion vessels. However, the traditional glass bottle is mainly designed to be a container for liquid, is not completely suitable for digestion of primary cells, and the traditional round bottom glass bottle limits sufficient contact between tissues and digestive enzymes, and repeatedly sucks digestive enzymes, so that on one hand, primary cell yield is reduced due to incomplete suction, and on the other hand, pollution probability in the digestion process is increased. The extraction of the tissue sample by the traditional digestion separation method has high requirements on technical operation, and the total primary cell yield can be influenced by the inexperienced technical operation and the overlong operation time. The unstable digestion separation extraction method finally wastes a great deal of manpower and financial resources to meet the experimental requirements, so that development of a digestion separation bottle special for primary cells is urgently needed. The applicant firstly designs an integrated primary cell digestion separation bottle according to the experimental operation process of extracting primary myocardial cells and adipose-derived mesenchymal stem cells for many years, summarizing experience and developing innovation.
Disclosure of Invention
The invention aims to solve the technical problems of the current digestion vessel and manufacture the experimental bottle which is convenient to operate and reasonable in structural design and is beneficial to digestion and separation of primary cells.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: the utility model provides an integral type primary cell digestion separating bottle, includes switch formula filter tip, aseptic protective sheath, malleation formula digestion bottle and digestion bottle support, switch formula filter tip one end detachable cover is equipped with aseptic protective sheath, the other end with malleation formula digestion bottle can dismantle and be connected, and is equipped with first filter screen, side at switch formula filter tip's inside and is equipped with rotary type check valve, be equipped with application of sample mouth and malleation mouth on the bottle body of malleation formula digestion bottle, and be equipped with the second filter screen at the one end that this malleation formula digestion bottle and switch formula filter tip are connected.
Further, the switch type filter tip comprises a cylinder and a cone which are connected, one end of the cylinder is provided with a screwed joint which is connected with the positive pressure type digestive tract, the other end of the cylinder is connected with a large opening of the cone, and the joint is provided with the first filter screen.
Furthermore, two circles of annular bosses used for fixing the first filter screen are arranged on the inner wall of the joint of the cylinder and the cone, and the specification of the first filter screen is 100 meshes or 200 meshes.
Further, the sterile protective sleeve is a transparent cylinder with one end sealed, and the opening section of the sterile protective sleeve is detachably combined with the switch type filter tip.
Further, the positive pressure type digestion bottle comprises a digestion bottle body and a digestion bottle neck, wherein the digestion bottle body is a cuboid bottle body, the digestion bottle neck is a multi-pyramid cone-shaped bottle neck, and the side face of the digestion bottle body is provided with volume scales. The digestion bottle bottleneck sets up in one side of digestion bottle body, the tip of digestion bottle bottleneck with the on-off type filter tip passes through threaded form and connects, is equipped with at the junction of digestion bottle bottleneck and digestion bottle body the second filter screen, be equipped with on the top surface of digestion bottle body the application of sample mouth is equipped with on the side the malleation mouth.
Furthermore, eight right-angle triangular brackets are arranged at the corners of the bottom surface of the digestion bottle body. The inner wall of the joint of the digestion bottle body and the digestion bottle bottleneck is provided with two circles of annular bosses for fixing a second filter screen, and the second filter screen has the specification of 35 meshes and can be used for blocking tissue agglomerates to block a filter tip screen opening so as to facilitate repeated digestion; the upper end of the sample adding port is a threaded port or a bayonet, usually a threaded port, with the diameter of 2cm, or a self-customized bayonet and any diameter; in addition, the positive pressure port for connecting the gas is usually 5mm in diameter, can be any diameter customized by self, and can be sealed by an integral or external cap.
The digestion bottle support comprises a support load base, a digestion bottle clamp and a support column, wherein one end of the support column is vertically fixed on the support load base, and the digestion bottle clamp is movably arranged on the support column.
The upper surface of support load base is equipped with a plurality of tube chute that is used for placing the centrifuging tube. The centrifuge tube can be selected to be 50ml in size and is mainly used for collecting digestive juice.
The beneficial effects of the invention are as follows:
the invention can provide 25cm according to the size of the tissue quantity 2 And 75cm 2 The digestion area of the cuboid bottle body with two specifications improves the contact area of tissues and digestive enzymes, and avoids the influence of adhesion and agglomeration of the tissues on digestion.
The invention can provide an integrated digestion and filtration process, effectively shortens a learning curve, shortens the operation time, and improves insufficient cell extraction caused by primary cell sedimentation with overlong operation time.
The invention ensures the convenience and high efficiency of primary cell extraction by a unique one-way switch control and positive pressure filtration mode. The sterile protective sleeve prevents the filter tip from contacting with the outside, and ensures sterility.
The digestion bottle body is provided with the 35-mesh iron filter screen, so that the filter tip is effectively prevented from being blocked by tissue blocks. The filter tip can provide different specifications of 200 meshes, 400 meshes and the like according to the primary cell extraction requirement, so that pollution is avoided, and the primary cell extraction purity is ensured.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The invention will be further described with reference to the drawings and examples.
FIG. 1 is a schematic diagram of the overall structure of the present invention.
Figure 2 is a schematic view of a switching filter and sterile protective sleeve of the present invention.
FIG. 3 is a schematic diagram of the structure of the positive pressure digestion vessel of the present invention.
FIG. 4 is a schematic diagram of the digestive flask support of the present invention.
Fig. 5 is a schematic diagram of the overall application of the present invention.
The reference numerals in the figures are: 1-a switching filter; 2-a sterile protective sleeve; 3-positive pressure digestion bottle; 4-digestive flask support; 5-a switch filter nipple; 6-a knob type one-way valve; 7-a first filter screen; 8-digesting the bottle body; 9-digesting bottle necks; 10-digesting the bottom surface of the bottle; 11-digestive flask sides; 12-a sample adding port; 13-positive pressure port; 14-digestive tract screw-threaded interface; 15-right angle triangular bracket; 16-a second filter screen; 17-a bracket load base; 18-digestion bottle clamp.
Detailed Description
For the purpose of making the technical solution and advantages of the present invention more apparent, the present invention will be further described with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
As shown in fig. 1 and 5, the integrated primary cell digestion and separation vessel comprises a switch filter 1, a sterile protective sleeve 2, a positive pressure digestion vessel 3 and a digestion vessel bracket 4.
As shown in fig. 2, the on-off filter tip comprises a cylinder and a cone which are connected, wherein one end of the cylinder, namely the large mouth end of the on-off filter tip, is provided with a screwed joint 5 for being connected with a positive pressure digestion bottle, and the small mouth end of the on-off filter tip is an outlet with the diameter of 2 mm. The rotary knob type one-way valve 6 is arranged on the cylinder, two circles of annular bosses used for fixing the first filter screen are arranged on the inner wall of the joint of the cylinder and the cone, and the specification of the first filter screen is 100 meshes or 200 meshes. In addition, the sterile protective sleeve is a transparent cylinder with one end sealed, and the opening section of the sterile protective sleeve is detachably combined with the switch type filter tip.
As shown in FIG. 3, the positive pressure digestion bottle consists of a rectangular digestion bottle body 8 and a multi-pyramid bottleneck 9, and can be customized to 25cm according to the size of the digestion tissue 2 And 75cm 2 The bottom surface 10 of the digestion bottle with the bottom area is provided with volume scales 11 on two sides, so that the digestion liquid can be conveniently metered. Three openings are arranged on the whole bottle body, one is a threaded opening right above, and the threaded opening is a sample adding port 12; the left side is a positive pressure port 13 which can be sealed by a heparin cap; on the right is an externally threaded opening, which is a digestive bottle threaded interface 14, connectable to the on-off filter 5. Four corners of the bottom surface of the digestion bottle are provided with 8 right triangle brackets 15. Cuboid digestive bottle body 8 and multi-edgeThe junction of the cone-shaped bottleneck 9 is provided with an annular boss for fixing the second filter screen 16. The second filter screen 16 is usually 35 mesh (any mesh can be selected according to the requirement), and is mainly used for blocking the filter mouth by the tissue mass, so as to facilitate the repeated digestion process.
Referring to fig. 4, the digestive bottle support comprises a support load base, a digestive bottle clamp and a support column, wherein one end of the support column is vertically fixed on the support load base, and the digestive bottle clamp is movably arranged on the support column. The upper surface of support load base is equipped with a plurality of tube chute that is used for placing the centrifuging tube. The centrifuge tube can be selected to be 50ml in size and is mainly used for collecting digestive juice.
The operation process of the integrated primary cell digestion and separation bottle comprises the following steps: 1. the positive pressure digester and the on-off filter were connected in a sterile bench according to fig. 1, the on-off filter being of the appropriate size (different mesh filters were chosen mainly with reference to the different primary cells). 2. And a sterile protective sleeve, a bottle cap and a sterile heparin cap are sleeved to ensure that all switches are in a closed state. 3. Cutting the tissue blocks to be digested into corresponding sizes according to the requirements, adding a proper amount of digestive enzymes, transferring the two together into a digestive flask, and adding the proper digestive enzymes according to the test requirements. 4. After the bottle cap is closed, ensuring that no effusion is completely moved into the incubator, and after the digestion time is over, the incubator is moved into the sterile operating table again. 5. The whole digestion vessel was placed on the digestion vessel holder as shown in FIG. 5, with a sterile 50ml centrifuge tube placed at the lower end. 6. After 1 minute of rest, remove sterile protective sheath, sterile heparin cap and unscrew switch, and digest would collect into centrifuge tube due to gravity. (optional steps: if filtration is to be faster and more complete, optionally positive pressure filtration is to be applied using a 50ml syringe connected to the positive pressure port.) 7. If repeated digestion is to be performed, the procedure can be repeated starting with 2 with the addition of the appropriate digestive enzymes, and the final digest is the desired primary cell suspension. The subsequent operations are operated according to the different primary cell extraction requirements.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It should be understood by those skilled in the art that the above embodiments do not limit the scope of the present invention in any way, and all technical solutions obtained by equivalent substitution and the like fall within the scope of the present invention.
The invention is not related in part to the same as or can be practiced with the prior art.
Claims (8)
1. An integral primary cell digestion separation bottle which is characterized in that: the disposable filter tip comprises a switch type filter tip, an aseptic protection sleeve, a positive pressure type digestion bottle and a digestion bottle support, wherein one end of the switch type filter tip is detachably sleeved with the aseptic protection sleeve, the other end of the switch type filter tip is detachably connected with the positive pressure type digestion bottle, a first filter screen is arranged in the switch type filter tip, a rotary one-way valve is arranged on the side face of the switch type filter tip, a sample adding port and a positive pressure port are arranged on the bottle body of the positive pressure type digestion bottle, and a second filter screen is arranged at one end, connected with the switch type filter tip, of the positive pressure type digestion bottle;
the switch type filter tip comprises a cylinder and a cone which are connected, one end of the cylinder is provided with a screwed joint used for being connected with the positive pressure type digestion bottle, the other end of the cylinder is connected with a large opening of the cone, and the joint is provided with the first filter screen;
the positive pressure type digestion bottle comprises a digestion bottle body and a digestion bottle bottleneck, the digestion bottle bottleneck is arranged on one side of the digestion bottle body, the end portion of the digestion bottle bottleneck is connected with the switch type filter tip in a threaded mode, the second filter screen is arranged at the joint of the digestion bottle bottleneck and the digestion bottle body, the sample adding port is arranged on the top surface of the digestion bottle body, and the positive pressure port is arranged on the side surface.
2. The integrated primary cell digestion separation flask of claim 1, wherein: two circles of annular bosses used for fixing the first filter screen are arranged on the inner wall of the joint of the cylinder and the cone, and the specification of the first filter screen is 100 meshes or 200 meshes.
3. The integrated primary cell digestion separation flask of claim 1, wherein: the sterile protective sleeve is a transparent cylinder with one end sealed, and the opening section of the sterile protective sleeve is detachably combined with the switch type filter tip.
4. The integrated primary cell digestion separation flask of claim 1, wherein: the digestion bottle body is a cuboid body, the digestion bottle neck is a multi-pyramid cone-shaped bottleneck, and the side face of the digestion bottle body is provided with volume scales.
5. The integrated primary cell digestion separation flask of claim 1, wherein: eight right-angle triangular brackets are arranged at the corners of the bottom surface of the digestion bottle body.
6. The integrated primary cell digestion separation flask of claim 1, wherein: two circles of annular bosses used for fixing a second filter screen are arranged on the inner wall of the joint of the digestive bottle body and the digestive bottle bottleneck, and the specification of the second filter screen is 35 meshes; the upper end of the sample adding port is a threaded port or a bayonet.
7. The integrated primary cell digestion separation flask of claim 1, wherein: the digestion bottle support comprises a support load base, a digestion bottle clamp and a support column, wherein one end of the support column is vertically fixed on the support load base, and the digestion bottle clamp is movably arranged on the support column.
8. The integrated primary cell digestion separation flask of claim 7, wherein: the upper surface of support load base is equipped with a plurality of tube chute that is used for placing the centrifuging tube.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811146980.0A CN108998378B (en) | 2018-09-29 | 2018-09-29 | Integrated primary cell digestion and separation bottle |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811146980.0A CN108998378B (en) | 2018-09-29 | 2018-09-29 | Integrated primary cell digestion and separation bottle |
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| Publication Number | Publication Date |
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| CN108998378A CN108998378A (en) | 2018-12-14 |
| CN108998378B true CN108998378B (en) | 2024-01-23 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201811146980.0A Active CN108998378B (en) | 2018-09-29 | 2018-09-29 | Integrated primary cell digestion and separation bottle |
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Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111363681A (en) * | 2020-03-19 | 2020-07-03 | 江苏省人民医院（南京医科大学第一附属医院） | Integrated primary cell climbing culture bottle |
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| CN201634683U (en) * | 2010-04-28 | 2010-11-17 | 侯绪伟 | Device for separating primary cells from tissue |
| CN204138673U (en) * | 2014-10-09 | 2015-02-04 | 沈阳澔源生物科技有限公司 | A kind of culture dish and culturing bottle dual-purpose apparatus |
| CN204138650U (en) * | 2014-10-09 | 2015-02-04 | 沈阳澔源生物科技有限公司 | A kind of autologous fat processing vessel |
| CN105586260A (en) * | 2016-03-11 | 2016-05-18 | 阜阳师范学院 | Layered cell coculturing device and method |
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| CN108179132A (en) * | 2018-03-14 | 2018-06-19 | 深圳海丽施生物医学材料科技有限公司 | A kind of isolated pancreatic islet device and method |
| CN208933385U (en) * | 2018-09-29 | 2019-06-04 | 南京广慈医疗科技有限公司 | Integral type primary cell digests separating bottle |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8309342B2 (en) * | 2007-12-04 | 2012-11-13 | Ingeneron, Inc. | Apparatus and methods for cell isolation |
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2018
- 2018-09-29 CN CN201811146980.0A patent/CN108998378B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005105977A1 (en) * | 2004-04-30 | 2005-11-10 | Ismatec Sa, Laboratoriumstechnik | Lid for bioreactor bottles and/or centrifugation tubes |
| CN201634683U (en) * | 2010-04-28 | 2010-11-17 | 侯绪伟 | Device for separating primary cells from tissue |
| CN204138673U (en) * | 2014-10-09 | 2015-02-04 | 沈阳澔源生物科技有限公司 | A kind of culture dish and culturing bottle dual-purpose apparatus |
| CN204138650U (en) * | 2014-10-09 | 2015-02-04 | 沈阳澔源生物科技有限公司 | A kind of autologous fat processing vessel |
| CN105586260A (en) * | 2016-03-11 | 2016-05-18 | 阜阳师范学院 | Layered cell coculturing device and method |
| CN205917261U (en) * | 2016-07-28 | 2017-02-01 | 中国人民解放军第四军医大学 | Primary cells digests quick separation device |
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| CN208933385U (en) * | 2018-09-29 | 2019-06-04 | 南京广慈医疗科技有限公司 | Integral type primary cell digests separating bottle |
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