CN116855380B - Stem cell expansion culture system and expansion culture method - Google Patents
Stem cell expansion culture system and expansion culture method Download PDFInfo
- Publication number
- CN116855380B CN116855380B CN202310829461.9A CN202310829461A CN116855380B CN 116855380 B CN116855380 B CN 116855380B CN 202310829461 A CN202310829461 A CN 202310829461A CN 116855380 B CN116855380 B CN 116855380B
- Authority
- CN
- China
- Prior art keywords
- storage tank
- culture
- liquid
- control unit
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 48
- 230000010261 cell growth Effects 0.000 title claims abstract description 36
- 238000012136 culture method Methods 0.000 title claims abstract description 15
- 239000007788 liquid Substances 0.000 claims abstract description 213
- 210000004027 cell Anatomy 0.000 claims abstract description 122
- 230000003321 amplification Effects 0.000 claims abstract description 39
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 39
- 230000008014 freezing Effects 0.000 claims abstract description 28
- 238000004113 cell culture Methods 0.000 claims abstract description 21
- 238000007710 freezing Methods 0.000 claims abstract description 17
- 239000001963 growth medium Substances 0.000 claims description 110
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 105
- 238000012546 transfer Methods 0.000 claims description 99
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 64
- 239000007853 buffer solution Substances 0.000 claims description 57
- 102000004142 Trypsin Human genes 0.000 claims description 43
- 108090000631 Trypsin Proteins 0.000 claims description 43
- 239000000243 solution Substances 0.000 claims description 43
- 239000012588 trypsin Substances 0.000 claims description 43
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 40
- 239000007921 spray Substances 0.000 claims description 34
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 24
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 21
- 239000000872 buffer Substances 0.000 claims description 20
- 229910052757 nitrogen Inorganic materials 0.000 claims description 20
- 239000006228 supernatant Substances 0.000 claims description 17
- 239000008367 deionised water Substances 0.000 claims description 16
- 229910021641 deionized water Inorganic materials 0.000 claims description 16
- 238000004820 blood count Methods 0.000 claims description 14
- 230000001276 controlling effect Effects 0.000 claims description 14
- 230000007246 mechanism Effects 0.000 claims description 14
- 238000005138 cryopreservation Methods 0.000 claims description 12
- 239000011550 stock solution Substances 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 10
- 238000005057 refrigeration Methods 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 8
- 210000004748 cultured cell Anatomy 0.000 claims description 7
- 230000001464 adherent effect Effects 0.000 claims description 6
- 239000006285 cell suspension Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000002699 waste material Substances 0.000 claims description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 239000002184 metal Substances 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 239000002910 solid waste Substances 0.000 claims description 3
- 238000007664 blowing Methods 0.000 claims description 2
- 230000012010 growth Effects 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 16
- 238000004321 preservation Methods 0.000 abstract description 9
- 238000010257 thawing Methods 0.000 abstract description 6
- 238000011084 recovery Methods 0.000 abstract description 5
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 3
- 239000006143 cell culture medium Substances 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- 238000012453 sprague-dawley rat model Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0236—Mechanical aspects
- A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
- A01N1/0252—Temperature controlling refrigerating apparatus, i.e. devices used to actively control the temperature of a designated internal volume, e.g. refrigerators, freeze-drying apparatus or liquid nitrogen baths
-
- 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
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/18—Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
-
- 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
- C12M23/52—Mobile; Means for transporting the apparatus
-
- 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
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/16—Vibrating; Shaking; Tilting
-
- 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
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
-
- 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
-
- 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
- C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
-
- 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
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
- C12M41/14—Incubators; Climatic chambers
-
- 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
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
-
- 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
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
-
- 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
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/48—Automatic or computerized control
-
- 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
- C12M45/00—Means for pre-treatment of biological substances
- C12M45/05—Means for pre-treatment of biological substances by centrifugation
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Sustainable Development (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Thermal Sciences (AREA)
- Physics & Mathematics (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Clinical Laboratory Science (AREA)
- Mechanical Engineering (AREA)
- Computer Hardware Design (AREA)
- Cell Biology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention provides a stem cell expansion culture system and an expansion culture method, wherein the stem cell expansion culture system comprises a control unit and an incubator; the incubator is divided into a liquid distribution area and a stem cell expansion culture area. The amplification culture method of the invention adopts a stem cell amplification culture system, thereby realizing full-automatic amplification culture of stem cells. The amplification culture system provided by the invention realizes automatic configuration of liquid, preservation of frozen solution, thawing recovery and the like in the cell amplification culture process, and utilizes a centrifugal tubular culture bottle, a pipette and a manipulator to directly take and put culture solution for cell culture, culture solution replacement in cell culture, and automatic freezing preservation after amplification culture in cell amplification culture, thereby realizing full-automatic flow operation of cell culture and avoiding pollution caused by manual operation.
Description
Technical Field
The invention relates to the field of stem cell culture, in particular to a stem cell expansion culture system and an expansion culture method.
Background
Stem cells have important applications in modern medicine and scientific research, but efficient and high-quality expansion of stem cells has been a difficult problem.
In conventional cell culture or expansion, cells are planted in a culture flask, a culture dish or the like, and after the cells proliferate to a certain density, the cells are digested, collected by centrifugation and passaged into a plurality of culture flasks for continuous culture. When the cells are cultured on a large scale, the culture medium needs to be replaced for about two days, and the cells need to be passaged for about four days, so that the daily maintenance operation needs to consume a large amount of time and labor cost; and the manual operation process is more, and the pollution is easy to cause.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a stem cell expansion culture system and an expansion culture method.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a stem cell expansion culture system comprising a control unit and an incubator;
the incubator is divided into a liquid preparation area and a stem cell expansion culture area, the incubator is provided with an ethanol spray gun, a manipulator, a liquid transfer gun, a plurality of ultraviolet lamps and a liquid transfer gun head box, the top of the incubator is provided with an XY axis sliding rail parallel to the top wall of the incubator, at least 3 spherical axes are arranged on the XY axis sliding rail, the ethanol spray gun, the manipulator and the liquid transfer gun are connected with the XY axis sliding rail through the spherical axes, and the ultraviolet lamps are arranged on the inner side of the incubator;
the liquid preparation area is provided with a deionized water liquid storage tank, a PBS buffer liquid storage tank, a dimethyl sulfoxide liquid storage tank, a trypsin liquid storage tank, a constant-temperature water bath tank A, a double-resistance liquid storage tank, a glutamine liquid storage tank and a frozen liquid storage tank, wherein a refrigeration sleeve is arranged on the outer side of the frozen liquid storage tank, the refrigeration sleeve controls the temperature of the frozen liquid storage tank to be-25 ℃ to-15 ℃, and the deionized water liquid storage tank is communicated with a PBS buffer liquid storage tank pipeline;
the constant temperature water bath box A is provided with a complete culture medium liquid storage tank, a basic culture medium storage tank and an FBS liquid storage tank, wherein the complete culture medium liquid storage tank is respectively communicated with the basic culture medium storage tank, the FBS liquid storage tank, the double-antibody liquid storage tank and the glutamine storage tank through pipelines, and the frozen storage liquid storage tank is respectively communicated with the dimethyl sulfoxide storage tank, the FBS liquid storage tank and the complete culture medium liquid storage tank through pipelines; the constant-temperature water bath box A is provided with an oscillating water bath tank, the complete culture medium liquid storage tank, the frozen liquid storage tank, the FBS liquid storage tank and the basic culture medium storage tank are detachably fixed in the oscillating water bath tank, the oscillating water bath tank of the constant-temperature water bath box A keeps constant temperature at any temperature of 0-10 ℃,
the stem cell expansion culture area is provided with a constant-temperature water bath box B, a cell incubator, a microscope, a blood cell counting plate, a vacuum cup, a screwing device, an inkjet marking device, a program cryopreservation device, a cryopreservation tube taking and placing box and a centrifuge, wherein the constant-temperature water bath box B is provided with a constant-temperature water tank, the constant-temperature water tank of the constant-temperature water bath box B keeps constant temperature at any temperature of 30-45 ℃, and the stem cell expansion culture area is also provided with an open PSB buffer solution transfer container, an open complete culture medium transfer container, an open cryopreservation solution transfer container and a trypsin solution transfer container; the open PSB buffer solution transfer container, the open complete culture medium transfer container, the open frozen stock solution transfer container and the trypsin solution transfer container are fixedly placed in a constant-temperature water tank of the constant-temperature water bath box B, the PBS buffer solution storage tank is communicated with the open PSB buffer solution transfer container pipeline, the complete culture medium liquid storage tank is communicated with the open complete culture medium transfer container pipeline, the frozen stock solution liquid storage tank is communicated with the open frozen stock solution transfer container pipeline, and the trypsin liquid storage tank is communicated with the trypsin solution transfer container pipeline;
a fixing frame is further arranged in the constant-temperature water tank of the constant-temperature water bath box B and used for fixing a culture bottle, the culture bottle is in a centrifugal tube shape, and the culture bottle can puncture a bottle cap; the centrifugal machine is provided with a centrifugal rotor, the centrifugal rotor is provided with a plurality of centrosymmetric centrifugal tube grooves, the centrifugal tube grooves can be used for accommodating centrifugal tubular culture bottles, the vacuum cups are communicated with an external liquid nitrogen tank through metal hoses, the screwing device is provided with a fixing mechanism and a screwing valve, the program freezing device is provided with a fixing groove, and the program freezing device controls the temperature in the fixing groove to be 0-minus 30 ℃;
the control unit programs all devices in the incubator.
The amplification culture system is provided with the ethanol spray gun, the manipulator, the liquid transfer gun, a plurality of ultraviolet lamps and a liquid transfer gun head box, and the ethanol spray gun, the manipulator and the liquid transfer gun are arranged on the XY axis sliding rail, so that the ethanol spray gun, the manipulator and the liquid transfer gun can move in parallel along the whole top wall plane of the culture box in the culture box, the ethanol spray gun, the manipulator and the liquid transfer gun are fixed through the spherical rotating shaft, 360-degree rotation can be carried out, the ethanol spray gun can spray ethanol for sterilization at all positions in the culture box, the manipulator can grasp a culture bottle, a freezing tube, a vacuum cup and the like under the control of the control unit, and the liquid transfer gun can absorb cell liquid, culture liquid and the like under the control of the control unit, so that automatic transfer of culture liquid components in the culture process is realized. And an ultraviolet lamp is arranged for disinfection.
The amplification culture system is provided with a deionized water storage tank, a PBS buffer storage tank, a dimethyl sulfoxide storage tank and a trypsin storage tank, a constant temperature water bath tank A, a double-resistance storage tank, a glutamine storage tank and a freezing storage liquid storage tank, and is respectively communicated with a basic culture medium storage tank, an FBS storage tank, a double-resistance storage tank and a glutamine storage tank through pipelines, so that automatic configuration of a complete culture medium is realized, the complete culture medium storage tank, the basic culture medium storage tank and the FBS storage tank are arranged in a constant temperature water tank of the constant temperature water bath tank A with the temperature controlled at 0-10 ℃, the cryopreservation of FBS and the basic culture medium is realized, the configured complete culture medium can be stored at 4 ℃, multiple freezing and thawing are avoided, the amplification culture system is provided with the freezing storage liquid storage tank, and the freezing storage liquid storage tank is respectively communicated with the dimethyl sulfoxide storage tank, the FBS storage tank and the full culture medium storage tank through pipelines, the freezing storage liquid raw material components can be conveyed through pipelines, and the freezing storage liquid can be automatically configured for standby at-20 ℃ through a refrigerating sleeve.
The expansion culture system is also provided with an open PSB buffer solution transfer container, an open complete culture medium transfer container, an open frozen stock solution transfer container and a trypsin solution transfer container in the stem cell expansion culture area; the open PSB buffer liquid transfer container, the open complete culture medium transfer container, the open frozen storage liquid transfer container and the trypsin solution transfer container are placed and fixed in the constant-temperature water tank of the constant-temperature water bath box B, the transfer container is communicated with a corresponding liquid storage tank pipeline, liquid to be used is input into the transfer device, the possibility of liquid pollution after configuration is avoided, the transfer container is utilized to automatically remove liquid by a liquid-transferring gun, the transfer container is arranged in the constant-temperature water bath box B with the temperature controlled between 30 and 45 ℃, the liquid to be taken can be preserved at 37 ℃ in advance, so that the liquid can be directly taken in the cell culture process, the efficiency is improved, the automation of the configuration and the taking of the culture liquid is realized, and the pollution caused by manual operation is reduced.
The expansion culture system is characterized in that a fixing frame is further arranged in the constant-temperature water tank B of the constant-temperature water bath box in the stem cell expansion culture area, the fixing frame is used for fixing a culture bottle, the culture bottle is in a centrifugal tube shape, and a bottle cover can be pierced by the culture bottle; the cell liquid, components of the cell culture medium, PBS buffer solution and the like can be removed through the control unit, the culture bottle is in a centrifugal tube shape, the PBS buffer solution can be added to clean cells after being directly grabbed by a manipulator controlled by the control unit to the centrifugal machine for centrifugation after being cultured, and the cells are resuspended by the culture medium, so that the automatic replacement of the culture solution in the cell culture process is realized, the above operation is realized in the centrifugal tube-shaped culture bottle, the complexity of liquid taking, placing and replacing is reduced, the automation of replacing the culture solution in the cell culture process is realized, and the pollution in the culture operation process is better reduced.
The amplification culture system monitors the cell condition through a microscope and a blood cell counting plate, measures the cell concentration, combines a centrifugal tubular culture bottle, a pipetting gun and a mechanical arm, automatically realizes the picking, placing and replacing of liquid in the amplification culture process in the whole process, directly centrifugates and cleans precipitated cells through the centrifugal tubular culture bottle after the amplification culture, directly adds cell cryopreservation liquid into a program cryopreservation device for freezing, and transfers the frozen cells to a liquid nitrogen tank for preservation after the frozen cells are frozen to minus 25 ℃.
The amplification culture system realizes automatic configuration of liquid, preservation of frozen solution, thawing recovery and the like in the cell amplification culture process, and utilizes a centrifugal tubular culture bottle, a pipette and a manipulator to directly take and put culture solution for cell culture, culture solution replacement in cell amplification culture and automatic freezing preservation after amplification culture, thereby realizing full-automatic flow operation of cell culture and avoiding pollution caused by manual operation.
Preferably, telescopic mechanisms are arranged between the ethanol spray gun and the spherical shaft, between the manipulator and the spherical shaft and between the liquid-transferring gun and the spherical shaft, one end of each telescopic mechanism is fixed on the spherical shaft, and the other end of each telescopic mechanism is fixed on the ethanol spray gun, the manipulator or the liquid-transferring gun.
The amplification culture system can adjust the heights of the ethanol spray gun, the manipulator and the liquid transferring gun, expands the moving range of the ethanol spray gun, the manipulator and the liquid transferring gun, and is convenient for taking and placing liquid, transferring liquid, grabbing a culture bottle, freezing a storage tube and the like.
Preferably, the telescopic structure is a cylinder, an oil cylinder or a telescopic spring.
Preferably, the open transfer container may be tubular, cup-shaped.
Preferably, the control unit controls the pipette gun, the ethanol spray gun and the manipulator to move along the XY axis sliding rail plane and rotate by 360 degrees along the spherical axis, the control unit controls the pipette gun, the ethanol spray gun and the manipulator to operate in a program, and the control unit controls the switch of the ultraviolet lamp.
Preferably, a waste liquid container and a solid waste container are arranged in the incubator.
Preferably, the control unit controls the constant temperature water bath box A and the constant temperature water bath box B to perform constant temperature adjustment and program operation; the control unit controls the program running of the centrifugal machine, the control unit controls the screwing device, the control unit controls the program freezing device, the control unit controls the microscope to monitor the cell condition in the culture bottle and feeds the monitoring image back to the control unit, the microscope calculates the cell concentration through the blood cell counting plate, and the control unit controls the inkjet marking device to mark.
Preferably, the control unit controls the cell incubator.
The invention also provides a stem cell expansion culture method, which comprises the following steps:
(1) The control unit starts the alcohol spray gun to sterilize, and simultaneously starts the ultraviolet lamp to sterilize for 20-40 minutes, and the alcohol volatilizes;
(2) Adding a proper amount of PBS buffer solution dry powder with a fixed amount into a PBS buffer solution storage tank, controlling deionized water in the deionized water storage tank by a control unit to quantitatively input into the PBS buffer solution storage tank to prepare PBS buffer solution, and adding a proper amount of trypsin, EDTA and deionized water into the trypsin buffer solution storage tank to prepare trypsin solution;
(3) The control unit starts the thermostatic water bath tank A to adjust the temperature of the oscillating water bath tank of the thermostatic water bath tank A to 4 ℃, the basic culture medium storage tank and the FBS liquid storage tank are respectively added with the basic culture medium and the FBS to recover at the temperature of 4 ℃, the double antibody and the glutamine are respectively added into the double antibody liquid storage tank and the glutamine storage tank to defrost, and after the double antibody and the glutamine are completely melted, the control unit controls the double antibody, the glutamine, the FBS and the basic culture medium to be input into the complete culture medium storage tank according to a set proportion and uniformly mixed;
(4) The control unit controls the resuscitated FBS, dimethyl sulfoxide and complete culture medium to be input into the frozen liquid storage tank from the corresponding storage according to the proportion and mixed uniformly, and the control unit starts the temperature of a refrigeration sleeve arranged at the outer side of the frozen liquid storage tank to be-20 ℃;
(5) The control unit controls the constant-temperature water bath box B to be opened, the temperature is regulated to 37 ℃ and kept constant, the control unit controls the external liquid nitrogen tank to input liquid nitrogen into the vacuum cup, controls the manipulator to take out the frozen storage tube for storing the primary stem cells stored in the liquid nitrogen, transfers the frozen storage tube into the vacuum cup for storing the liquid nitrogen, and then transfers the frozen storage tube into the constant-temperature water tank of the constant-temperature water bath box B to defrost the frozen storage liquid of the primary cells;
(6) The control unit controls the PBS buffer solution storage tank, the PBS buffer solution in the complete culture medium storage tank and the complete culture medium to be respectively conveyed to the PSB buffer solution transfer container and the open complete culture medium transfer container to be resuscitated at the constant temperature of 37 ℃;
(7) The control unit controls the manipulator and the liquid transferring gun to absorb the cell liquid, the complete culture medium in the open complete culture medium transferring container is transferred to the culture flask, the complete culture medium is gently and evenly mixed, the PBS buffer liquid with equal mass in the PSB buffer liquid transferring container is absorbed into the other culture flask, the control unit controls the screwing device to screw the bottle cap of the culture flask, the manipulator moves the bottle cap into the centrifugal machine, the control unit controls the centrifugal machine to centrifuge for 5-10 minutes at the speed of 400-800 revolutions per minute, the mechanical hand is used for taking out the culture flask with the cell, the screwing device is controlled to open the bottle cap, the supernatant is discarded, a proper amount of culture medium is absorbed for re-suspending the precipitated primary stem cells, and after calculating the cell concentration under a microscope, the liquid transferring gun absorbs the quantitative complete culture medium to adjust the re-suspended cells to a proper concentration;
(8) After the control unit controls the inkjet marking device to mark the culture bottle obtained in the step (7), the manipulator places the marked culture bottle into CO 2 Culturing in a constant temperature cell incubator;
(9) The culture medium is replaced at 45-51 hours of culture interval of the culture bottle;
(10) When the growth proportion of the cells reaches 75% -90% through observation of a microscope, carrying out passage amplification culture;
(11) Centrifuging a culture bottle after passage expansion culture, discarding supernatant, adding PBS buffer solution to moisten and wash adherent cells, adding trypsin solution, putting the cells into a cell incubator to digest for 1-3min, stopping the cells with a complete culture medium in time after complete digestion, centrifuging, discarding supernatant, adding frozen stock solution, transferring the frozen stock solution into a frozen stock tube, marking the frozen stock tube by an ink-jet marking device, putting the frozen stock tube into a program frozen stock device to freeze the frozen stock tube to below-20 ℃, and transferring the frozen stock tube into a liquid nitrogen tank for storage.
Preferably, in the step (9), the control unit controls the pipette to suck PBS buffer solution with medium quality in the culture flask into another culture flask, the control unit controls the screwing device to screw the bottle cap of the culture flask, then the cell culture flask and the buffer solution culture flask are moved into the centrifuge by the manipulator, the control unit controls the centrifuge to centrifuge for 5-10 minutes at the speed of 400-800 rpm, the bottle cap of the cell placing culture flask is opened by the manipulator, the supernatant is discarded after the bottle cap of the cell placing culture flask is opened by the manipulator, a proper amount of culture medium is sucked for re-suspending the precipitated stem cells, and after the cell concentration is calculated under a microscope by the blood cell counting plate, the pipette sucks the quantitative complete culture medium and adjusts the re-suspended cells to a proper concentration.
Preferably, in the step (10), the control unit controls the trypsin solution in the trypsin liquid storage tank to be resuscitated at a constant temperature of 37 ℃ in the trypsin solution transferring container; centrifuging the culture bottle containing the cultured cells after culturing in the step (9) to remove supernatant, soaking and washing the cells by using PBS buffer solution, then, using a liquid transfer gun to absorb trypsin solution into the culture bottle containing the cultured cells, covering a cover, putting the culture bottle containing the cultured cells into a cell incubator to digest for 1-3min, taking out the culture bottle, using the liquid transfer gun to absorb a proper amount of complete culture medium in a complete culture medium transfer container into the culture bottle, using a liquid transfer device to blow the adherent cells remained on the surface of the bottle into the culture medium, absorbing PBS buffer solution with equal mass in the PSB buffer solution transfer container into another culture bottle, controlling a screwing device to screw a bottle cover by using a control unit, then, using a manipulator to move the manipulator into a centrifuge, controlling the centrifuge to centrifuge at a speed of 400-800 revolutions per minute for 5-10 min, using the manipulator to take out the culture bottle with the cell put into the screwing device to digest the supernatant after opening the cover, absorbing the complete culture medium into the culture bottle with a sedimentation cell completely, using a blood cell count plate to calculate the cell concentration under a microscope, after the cell count plate is sub-pumped into a proper concentration, sub-packaging the cells, using the control unit to spray the cell count into a bottle after marking, and placing a CO label in the control unit, and placing the culture bottle after the control unit into a bottle to print the bottle after marking 2 And subculturing the cells in a constant temperature cell incubator in a ratio of 1:1.5-1:3.
The stem cell expansion culture method is written into the control unit through a programming language, and the control unit automatically controls the operation of the method, so that the stem cell full-automatic expansion culture is realized.
The invention has the beneficial effects that: the invention provides a stem cell expansion culture system and an expansion culture method, wherein the expansion culture system provided by the invention realizes that an ethanol spray gun can spray ethanol for disinfection at all positions in an incubator, a manipulator can grasp a culture bottle, a freezing tube, a thermos cup and the like under the control of a control unit, and a liquid-transferring gun can absorb cell liquid, culture liquid and the like under the control of the control unit, so that automatic transfer of culture liquid components in the culture process is realized. And an ultraviolet lamp is arranged for disinfection. The invention realizes automatic configuration of complete culture medium, realizes recovery of cryopreserved FBS and basic culture medium, and the configured complete culture medium can be stored at 4 ℃ to avoid repeated freezing and thawing. The amplification culture system is communicated with the corresponding liquid storage tank through the open transfer container, the liquid to be used is input into the transfer device, the possibility of pollution of the configured liquid is avoided, the transfer container is utilized to realize automatic liquid transferring by the liquid transferring gun, the transfer container is arranged in the constant-temperature water bath box B with the temperature controlled at 30-45 ℃, the liquid to be taken can be preserved at 37 ℃ in advance, so that the liquid to be taken can be taken directly in the cell culture process, the efficiency is improved, the automation of the configuration and the taking of the culture liquid is realized, and the pollution of manual operation is reduced. The amplification culture system can remove cell liquid, components of a cell culture medium, PBS buffer solution and the like through the control unit, the culture bottle is in a centrifugal tube shape, and can be directly grasped by a manipulator controlled by the control unit after culture to a centrifuge for centrifugation, then PBS buffer solution is added to clean cells, and the culture medium is used for resuspension of the cells, so that the automatic replacement of the culture liquid in the cell culture process is realized, the operations are realized in the centrifugal tube-shaped culture bottle, the complexity of liquid taking, placing and replacing is reduced, the automation of the replacement of the culture liquid in the cell culture process is realized, and the pollution in the culture operation process is better reduced. The amplification culture system monitors the cell condition through a microscope and a blood cell counting plate, measures the cell concentration, combines a centrifugal tubular culture bottle, a pipetting gun and a mechanical arm, realizes the picking, placing and replacing of liquid in the amplification culture process in an automatic whole process, directly centrifugates and cleans precipitated cells through the centrifugal tubular culture bottle after the amplification culture, directly adds cell cryopreservation liquid, freezes in a program cryopreservation device, and transfers the frozen cells to a liquid nitrogen tank for preservation after the frozen cells are frozen to minus 25 ℃. The amplification culture system provided by the invention realizes automatic configuration of liquid, preservation of frozen solution, thawing recovery and the like in the cell amplification culture process, and utilizes a centrifugal tubular culture bottle, a pipette and a manipulator to directly take and put culture solution for cell culture, culture solution replacement in cell culture, and automatic freezing preservation after amplification culture in cell amplification culture, thereby realizing full-automatic flow operation of cell culture and avoiding pollution caused by manual operation.
Drawings
FIG. 1 is a schematic diagram of a stem cell expansion culture system according to an embodiment of the invention.
FIG. 2 is a schematic diagram showing the structure of a stem cell expansion culture system according to an embodiment of the present invention from a top view along section A-A.
The device comprises a control unit 1, a circuit 3, an incubator 4, an XY axis sliding rail 5, an ethanol spray gun 6, a manipulator 7, a pipetting gun 8, a screwing device 9, an inkjet marking device 10, a spherical axis 11, a telescopic structure 12, an ultraviolet lamp 13, an oscillating water bath tank of the constant temperature water bath tank A14, the constant temperature water bath tank A15, a complete culture medium liquid storage tank 16, a basal culture medium storage tank 17, an FBS liquid storage tank 18, a frozen storage liquid tank, a constant temperature water tank 19, a centrifuge 20, a constant temperature water tank of the constant temperature water bath tank B21, the constant temperature water bath tank B22, an open PSB buffer medium transfer container 23, an open complete culture medium transfer container 24, an open frozen storage liquid transfer container 25, a trypsin solution transfer container 26, an ion water storage tank 27, a PBS buffer liquid storage tank 28, a dimethyl sulfoxide storage tank 29, a trypsin liquid storage tank 30, a double-resistant liquid tank 31, a glutamine storage tank 32, a waste liquid container 33, a cell incubation tank 34, a microscope 35, a frozen storage cup 36, a storage tube 37 and a heat preservation program taking device.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples.
Example 1
As the stem cell expansion culture system provided by the embodiment of the invention, the stem cell expansion culture system comprises a control unit (1) and an incubator (3);
the incubator is divided into a liquid preparation area and a stem cell expansion culture area, the incubator (3) is provided with an ethanol spray gun (5), a manipulator (6), a liquid-transferring gun (7), a plurality of ultraviolet lamps (12) and a liquid-transferring gun head box, the top of the incubator is provided with an XY axis sliding rail (4) parallel to the top wall of the incubator, at least 3 spherical axes (10) are arranged on the XY axis sliding rail (4), the ethanol spray gun (5), the manipulator (6) and the liquid-transferring gun (7) are connected with the XY axis sliding rail (4) through the spherical axes (10), and the ultraviolet lamps (12) are arranged on the inner side of the incubator;
the liquid preparation area is provided with a deionized water liquid storage tank (26), a PBS buffer liquid storage tank (27), a dimethyl sulfoxide storage tank (28), a trypsin liquid storage tank (29), a constant-temperature water bath tank A (14), a double-antibody liquid storage tank (30), a glutamine storage tank (31) and a frozen liquid storage tank (18), the outer side of the frozen liquid storage tank (18) is provided with a refrigeration sleeve, the refrigeration sleeve controls the temperature of the frozen liquid storage tank (18) to be-25 ℃ to-15 ℃, and the deionized water liquid storage tank (26) is communicated with a pipeline of the PBS buffer liquid storage tank (27);
the constant temperature water bath box A (14) is provided with a complete culture medium liquid storage tank (15), a basic culture medium storage tank (16) and an FBS liquid storage tank (17), wherein the complete culture medium liquid storage tank (15) is respectively communicated with the basic culture medium storage tank (16), the FBS liquid storage tank (17), the double-antibody liquid storage tank (30) and the glutamine storage tank (31) through pipelines, and the frozen liquid storage tank (18) is respectively communicated with the dimethyl sulfoxide storage tank (28), the FBS liquid storage tank (17) and the complete culture medium liquid storage tank (15) through pipelines; the constant-temperature water bath box A (14) is provided with an oscillating water bath tank (13), a complete culture medium liquid storage tank (15), a frozen liquid storage tank (18), an FBS liquid storage tank (17) and a basic culture medium storage tank (16) are detachably fixed in the oscillating water bath tank (13), and the oscillating water bath tank (13) of the constant-temperature water bath box A (14) keeps constant temperature at any temperature of 0-10 ℃;
the stem cell expansion culture area is provided with a constant-temperature water bath box B (21), a cell incubator (33), a microscope (34), a blood cell counting plate, a vacuum cup (35), a screwing device (8), an inkjet marking device (9), a program freezing device (37) and a freezing tube taking and placing box (36), the constant-temperature water bath box B (21) is provided with a constant-temperature water tank (20), the constant-temperature water tank (20) of the constant-temperature water bath box B (21) keeps constant at any temperature of 30-45 ℃, and the stem cell expansion culture area is also provided with an open PSB buffer medium transfer container (22), an open complete culture medium transfer container (23), an open freezing liquid transfer container (24) and a trypsin solution transfer container (25); an open PSB buffer medium transfer container (22), an open complete culture medium transfer container (23), an open cryopreservation liquid transfer container (24) and a trypsin solution transfer container (25) are placed and fixed in a constant-temperature water tank (20) of a constant-temperature water bath box B (21), a PBS buffer liquid storage tank (27) is communicated with the open PSB buffer medium transfer container (22) through a pipeline, a complete culture medium liquid storage tank (15) is communicated with the open complete culture medium transfer container (23) through a pipeline, a cryopreservation liquid storage tank (18) is communicated with the open cryopreservation liquid transfer container (24) through a pipeline, and a trypsin liquid storage tank (29) is communicated with the trypsin solution transfer container (25) through a pipeline;
the screwing device (8) and the inkjet marking device (8) are respectively fixed on the XY-axis sliding rail (4) through a telescopic mechanism (11) and a spherical axis (10);
a fixing frame is also arranged in the constant-temperature water tank (20) of the constant-temperature water bath box B and is used for fixing a culture bottle which is in a centrifugal tube shape, and the culture bottle can puncture a bottle cap; the centrifugal machine (19) is provided with a centrifugal rotor, the centrifugal rotor is provided with a plurality of centrosymmetric centrifugal tube grooves, the centrifugal tube grooves can be used for accommodating centrifugal tubular culture bottles, the vacuum cup (35) is communicated with an external liquid nitrogen tank through a metal hose, the screwing device (8) is provided with a fixing mechanism and a screwing valve, the program freezing device (37) is provided with a fixing groove, and the temperature in the fixing groove is controlled to be 0-minus 30 ℃ by the program freezing device (37);
telescopic mechanisms (11) are arranged between the ethanol spray gun (5) and the spherical shaft (10), between the manipulator (6) and the spherical shaft (10) and between the liquid-transferring gun (7) and the spherical shaft (10), one end of each telescopic mechanism is fixed on the spherical shaft, and the other end of each telescopic mechanism is fixed on the ethanol spray gun (5), the manipulator (6) or the liquid-transferring gun (7).
The telescopic structure (11) is an air cylinder, the air cylinder is controlled to stretch by the control unit (1), and the open transfer container can be tubular or cup-shaped.
The control unit (1) realizes programming control on the structure in the incubator (3) through the circuit (2).
The control unit (1) controls the pipetting gun (5), the ethanol spray gun (6) and the manipulator (7) to move along the plane of the XY-axis sliding rail (4) and rotate by 360 degrees through the spherical axis (10), the control unit (1) controls the pipetting gun (5), the ethanol spray gun (6) and the manipulator (7) to operate in a program mode, and the control unit (1) controls the switch of the ultraviolet lamp (12).
A waste liquid container (32) and a solid waste container are arranged in the incubator (3).
The control unit (1) controls the constant temperature water bath box A (14) and the constant temperature water bath box B (21) to perform constant temperature adjustment and program operation; the control unit (1) controls the program running of the centrifugal machine (19), the control unit (1) controls the screwing device (8), the control unit (1) controls the program freezing device (37), the control unit (1) controls the microscope (34) to monitor the cell condition in the culture flask and feed the monitoring image back to the control unit, the microscope (34) calculates the cell concentration through the blood cell counting plate, and the control unit (1) controls the inkjet marking device (9) to mark.
The control unit (1) controls the cell incubator (33).
As a stem cell expansion culture method of an embodiment of the present invention, the expansion culture method of the present embodiment is performed using the stem cell expansion culture system of the present embodiment, the method comprising the steps of:
(1) The control unit starts the alcohol spray gun to sterilize, and simultaneously starts the ultraviolet lamp to sterilize for 30 minutes, and the alcohol volatilizes;
(2) Adding a proper amount of PBS buffer solution dry powder with a fixed amount into a PBS buffer solution storage tank, controlling deionized water in the deionized water storage tank by a control unit to quantitatively input into the PBS buffer solution storage tank to prepare PBS buffer solution, and adding a proper amount of trypsin, EDTA and deionized water into the trypsin buffer solution storage tank to prepare trypsin solution;
(3) The control unit starts the thermostatic water bath tank A to adjust the temperature of the oscillating water bath tank of the thermostatic water bath tank A to 4 ℃, the basic culture medium storage tank and the FBS liquid storage tank are respectively added with the basic culture medium and the FBS to recover at the temperature of 4 ℃, the double antibody and the glutamine are respectively added into the double antibody liquid storage tank and the glutamine storage tank to defrost, and after the double antibody and the glutamine are completely melted, the control unit controls the double antibody, the glutamine, the FBS and the basic culture medium to be input into the complete culture medium storage tank according to a set proportion and uniformly mixed;
(4) The control unit controls the resuscitated FBS, dimethyl sulfoxide and complete culture medium to be input into the frozen liquid storage tank from the corresponding storage according to the proportion and mixed uniformly, and the control unit starts the temperature of a refrigeration sleeve arranged at the outer side of the frozen liquid storage tank to be-20 ℃;
(5) The control unit controls the constant-temperature water bath box B to be opened, the temperature is regulated to 37 ℃ and kept constant, the control unit controls the external liquid nitrogen tank to input liquid nitrogen into the vacuum cup, controls the manipulator to take out the frozen storage tube for storing the primary stem cells stored in the liquid nitrogen, transfers the frozen storage tube into the vacuum cup for storing the liquid nitrogen, and then transfers the frozen storage tube into the constant-temperature water tank of the constant-temperature water bath box B to defrost the frozen storage liquid of the primary cells;
(6) The control unit controls the PBS buffer solution storage tank, the PBS buffer solution in the complete culture medium storage tank and the complete culture medium to be respectively conveyed to the PSB buffer solution transfer container and the open complete culture medium transfer container to be resuscitated at the constant temperature of 37 ℃;
(7) The control unit controls the manipulator and the liquid transferring gun to absorb the cell liquid, the complete culture medium in the open complete culture medium transferring container is transferred to the culture flask, the complete culture medium is gently and evenly mixed, the PBS buffer liquid with equal mass in the PSB buffer liquid transferring container is absorbed into the other culture flask, the control unit controls the screwing device to screw the bottle cap of the culture flask, the manipulator moves the bottle cap into the centrifuge, the control unit controls the centrifuge to centrifuge for 6 minutes at the speed of 500 revolutions per minute, the manipulator is used for taking out the culture flask with the cells, the screwing device is controlled to open the bottle cap, the supernatant is discarded, a proper amount of culture medium is absorbed for re-suspending the precipitated primary stem cells, and after calculating the cell concentration under a microscope, the liquid transferring gun absorbs the quantitative complete culture medium to adjust the re-suspended cells to a proper concentration;
(8) After the control unit controls the inkjet marking device to mark the culture bottle obtained in the step (7), the manipulator places the marked culture bottle into CO 2 Culturing in a constant temperature cell incubator;
(9) The control unit controls the liquid transferring gun to absorb PBS buffer solution with medium quality in the culture bottle to the other culture bottle, after the control unit controls the screwing device to screw the bottle cap of the culture bottle, the cell culture bottle and the buffer solution culture bottle are moved into the centrifugal machine by the mechanical arm, the control unit controls the centrifugal machine to centrifuge for 6 minutes at the speed of 500 revolutions per minute, the bottle cap of the cell placing culture bottle is opened by the mechanical arm to control the screwing device, the supernatant is discarded, a proper amount of culture medium is absorbed for resuspension of precipitated stem cells, and after the cell concentration is calculated under a microscope by the blood cell counting plate, the quantitative complete culture medium is absorbed by the liquid transferring gun, and the resuspension of the cells is adjusted to a proper concentration;
(10) The control unit controls trypsin solution in the trypsin liquid storage tank to be resuscitated at a constant temperature of 37 ℃ in the trypsin solution transfer container; centrifuging the culture bottle containing the cultured cells after culturing in the step (9) to remove supernatant, soaking the cells in PBS buffer solution, then removing the cells in the PBS buffer solution, covering a cover in the culture bottle containing the cultured cells by a liquid-transferring gun, putting the culture bottle into a cell incubator to digest for 2min, taking out the culture bottle, sucking a proper amount of complete medium in a complete medium transfer container into the culture bottle by the liquid-transferring gun, blowing the adherent cells remained on the surface of the bottle into the culture medium by a liquid-transferring gun, sucking the PBS buffer solution with equal mass in the PSB buffer solution transfer container into another culture bottle, controlling the bottle cover of the culture bottle by a screwing device by a control unit, moving the PBS buffer solution into a centrifuge by a manipulator, controlling the centrifuge to centrifuge for 6 min at a speed of 500 rpm, taking out the culture bottle containing the cells by the manipulator, controlling the bottle cover of the screwing device to discard the supernatant, sucking the complete medium into the culture bottle of the sedimentation cells, completely mixing the cells into single cell suspension by a blood cell counting plate under a microscope, calculating the cell concentration, after adjusting the cell suspension to a proper concentration, subpackaging the cell suspension in the culture bottle, controlling the ink bottle to control the marker, and spraying the cell suspension into the culture bottle after marking the CO suspension by the control unit, and controlling the bottle to spray the marker to be placed into the culture bottle 2 Subculturing in a constant temperature cell incubator at a ratio of 1:2;
(11) Centrifuging a culture bottle after passage expansion culture, discarding supernatant, adding PBS buffer solution to moisten and wash adherent cells, adding trypsin solution, putting the cells into a cell incubator to digest for 1-3min, stopping the cells with a complete culture medium in time after complete digestion, centrifuging, discarding supernatant, adding frozen stock solution, transferring the frozen stock solution into a frozen stock tube, marking the frozen stock tube by an ink-jet marking device, putting the frozen stock tube into a program frozen stock device to freeze the frozen stock tube to below-20 ℃, and transferring the frozen stock tube into a liquid nitrogen tank for storage.
Experimental example
1. Expansion culture was performed using Sprague-Dawley (SD) rat primary BM-MSCs (passaged to 2 nd generation cryopreservation) as a sample.
2. The amplification culture system and the amplification culture method of example 1 were used for subculture, and the third generation cells were cultured in 30 flasks in one amplification culture cycle.
Three cycles of amplification culture were cultured as parallel experiments, and after culturing, the number of contaminating cells was monitored using a flask as a counting unit.
3. According to the amplification culture procedure of example 1, all of the cells were manually subjected to passaged amplification culture, and the cells were subjected to amplification culture to the third generation in one amplification culture cycle, and the third generation cells were cultured in 30 flasks.
Three cycles of amplification culture were cultured as parallel experiments, and after culturing, the number of contaminating cells was monitored using a flask as a counting unit.
The experimental results are shown in table 1.
TABLE 1 contamination ratio of amplification culture
As is clear from the results of Table 1, the cell expansion culture method of example 1 was used, only one flask was contaminated during one cycle, and in the manual operation, 5.7 flasks were contaminated on average during 30 flasks per cycle, and therefore, the stem cell expansion culture system and the stem cell expansion culture method of example were realized in automatic configuration of the liquid used in the cell expansion culture process, preservation of the frozen solution, thawing recovery, and the like, and the culture solution was directly taken and placed by using a centrifuge tube type flask, a pipette, and a manipulator for cell culture, culture solution replacement in cell expansion culture, automatic cryopreservation after expansion culture, and realization of the operation of the full-automatic flow of cell culture, and avoidance of contamination caused in the manual operation.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (2)
1. A stem cell expansion culture system, which is characterized by comprising a control unit and an incubator;
the incubator is divided into a liquid preparation area and a stem cell expansion culture area, the incubator is provided with an ethanol spray gun, a manipulator, a liquid transfer gun, a plurality of ultraviolet lamps and a liquid transfer gun head box, the top of the incubator is provided with an XY axis sliding rail parallel to the top wall of the incubator, at least 3 spherical axes are arranged on the XY axis sliding rail, the ethanol spray gun, the manipulator and the liquid transfer gun are connected with the XY axis sliding rail through the spherical axes, and the ultraviolet lamps are arranged on the inner side of the incubator;
the liquid preparation area is provided with a deionized water liquid storage tank, a PBS buffer liquid storage tank, a dimethyl sulfoxide liquid storage tank, a trypsin liquid storage tank, a constant-temperature water bath tank A, a double-resistance liquid storage tank, a glutamine liquid storage tank and a frozen liquid storage tank, wherein a refrigeration sleeve is arranged on the outer side of the frozen liquid storage tank, the refrigeration sleeve controls the temperature of the frozen liquid storage tank to be minus 25 ℃ to minus 15 ℃, and the deionized water liquid storage tank is communicated with a pipeline of the PBS buffer liquid storage tank;
the constant temperature water bath box A is provided with a complete culture medium liquid storage tank, a basic culture medium storage tank and an FBS liquid storage tank, wherein the complete culture medium liquid storage tank is respectively communicated with the basic culture medium storage tank, the FBS liquid storage tank, the double-antibody liquid storage tank and the glutamine storage tank through pipelines, and the frozen storage liquid storage tank is respectively communicated with the dimethyl sulfoxide storage tank, the FBS liquid storage tank and the complete culture medium liquid storage tank through pipelines; the constant-temperature water bath box A is provided with an oscillating water bath tank, the complete culture medium liquid storage tank, the frozen liquid storage tank, the FBS liquid storage tank and the basic culture medium storage tank are detachably fixed in the oscillating water bath tank, the oscillating water bath tank of the constant-temperature water bath box A keeps constant temperature at any temperature of 0-10 ℃,
the stem cell expansion culture area is provided with a constant-temperature water bath box B, a cell incubator, a microscope, a blood cell counting plate, a vacuum cup, a screwing device, an inkjet marking device, a program cryopreservation device, a cryopreservation tube taking and placing box and a centrifuge, wherein the constant-temperature water bath box B is provided with a constant-temperature water tank, the constant-temperature water tank of the constant-temperature water bath box B keeps constant temperature at any temperature of 30-45 ℃, and the stem cell expansion culture area is also provided with an open PSB buffer solution transfer container, an open complete culture medium transfer container, an open cryopreservation solution transfer container and a trypsin solution transfer container; the open PSB buffer solution transfer container, the open complete culture medium transfer container, the open frozen stock solution transfer container and the trypsin solution transfer container are fixedly placed in a constant-temperature water tank of the constant-temperature water bath box B, the PBS buffer solution storage tank is communicated with the open PSB buffer solution transfer container pipeline, the complete culture medium liquid storage tank is communicated with the open complete culture medium transfer container pipeline, the frozen stock solution liquid storage tank is communicated with the open frozen stock solution transfer container pipeline, and the trypsin liquid storage tank is communicated with the trypsin solution transfer container pipeline;
a fixing frame is further arranged in the constant-temperature water tank of the constant-temperature water bath box B and used for fixing a culture bottle, the culture bottle is in a centrifugal tube shape, and the culture bottle can puncture a bottle cap; the centrifugal machine comprises a centrifugal rotor, a screw device and a program freezing device, wherein the centrifugal rotor is provided with a plurality of centrosymmetric centrifugal tube grooves, the centrifugal tube grooves can be used for accommodating centrifugal tubular culture bottles, the vacuum cups are communicated with an external liquid nitrogen tank through metal hoses, the screw device is provided with a fixing mechanism and a screw valve, the program freezing device is provided with a fixing groove, and the program freezing device controls the temperature in the fixing groove to be 0-30 ℃;
the control unit program controls all devices in the incubator;
telescopic mechanisms are arranged between the ethanol spray gun and the spherical shaft, between the manipulator and the spherical shaft and between the liquid-transferring gun and the spherical shaft, one end of each telescopic mechanism is fixed on the spherical shaft, and the other end of each telescopic mechanism is fixed on the ethanol spray gun, the manipulator or the liquid-transferring gun;
the telescopic mechanism is an air cylinder, an oil cylinder or a telescopic spring;
the control unit controls the pipette gun, the ethanol spray gun and the manipulator to move along the XY axis sliding rail plane and rotate by 360 degrees on the spherical axis, the control unit controls the pipette gun, the ethanol spray gun and the manipulator to operate in a program, and the control unit controls the switch of the ultraviolet lamp;
a waste liquid container and a solid waste container are arranged in the incubator;
the control unit controls the constant-temperature water bath box A and the constant-temperature water bath box B to perform constant-temperature adjustment and program operation; the control unit controls the program running of the centrifugal machine, the control unit controls the screwing device, the control unit controls the program freezing device, the control unit controls the microscope to monitor the cell condition in the culture bottle and feeds a monitoring image back to the control unit, the microscope calculates the cell concentration through the blood cell counting plate, and the control unit controls the inkjet marking device to mark;
the control unit controls the cell incubator.
2. A stem cell expansion culture method, characterized in that the stem cell expansion culture system according to claim 1 is used, comprising the steps of:
(1) The control unit starts the alcohol spray gun to sterilize, and simultaneously starts the ultraviolet lamp to sterilize for 20-40 minutes, and the alcohol volatilizes;
(2) Adding a proper amount of PBS buffer solution dry powder with a fixed amount into a PBS buffer solution storage tank, controlling deionized water in the deionized water storage tank by a control unit to quantitatively input into the PBS buffer solution storage tank to prepare PBS buffer solution, and adding a proper amount of trypsin, EDTA and deionized water into the trypsin buffer solution storage tank to prepare trypsin solution;
(3) The control unit starts the thermostatic water bath tank A to adjust the temperature of the oscillating water bath tank of the thermostatic water bath tank A to 4 ℃, the basic culture medium storage tank and the FBS liquid storage tank are respectively added with the basic culture medium and the FBS to recover at the temperature of 4 ℃, the double antibody and the glutamine are respectively added into the double antibody liquid storage tank and the glutamine storage tank to defrost, and after the double antibody and the glutamine are completely melted, the control unit controls the double antibody, the glutamine, the FBS and the basic culture medium to be input into the complete culture medium storage tank according to a set proportion and uniformly mixed;
(4) The control unit controls the resuscitated FBS, dimethyl sulfoxide and complete culture medium to be input into the frozen liquid storage tank from the corresponding storage according to the proportion and mixed uniformly, and the control unit starts the temperature of a refrigeration sleeve arranged at the outer side of the frozen liquid storage tank to be-20 ℃;
(5) The control unit controls the constant-temperature water bath box B to be opened, the temperature is regulated to 37 ℃ and kept constant, the control unit controls the external liquid nitrogen tank to input liquid nitrogen into the vacuum cup, controls the manipulator to take out the frozen storage tube for storing the primary stem cells stored in the liquid nitrogen, transfers the frozen storage tube into the vacuum cup for storing the liquid nitrogen, and then transfers the frozen storage tube into the constant-temperature water tank of the constant-temperature water bath box B to defrost the frozen storage liquid of the primary cells;
(6) The control unit controls the PBS buffer solution storage tank, the PBS buffer solution in the complete culture medium storage tank and the complete culture medium to be respectively conveyed to the PSB buffer solution transfer container and the open complete culture medium transfer container to be resuscitated at the constant temperature of 37 ℃;
(7) The control unit controls the manipulator and the liquid transferring gun to absorb the cell liquid, the complete culture medium in the open complete culture medium transfer container is transferred to the culture flask, the complete culture medium is gently and evenly mixed, the PBS buffer liquid with equal mass in the PSB buffer liquid transfer container is absorbed into the other culture flask, the control unit controls the screwing device to screw the bottle cap of the culture flask, the manipulator moves the bottle cap into the centrifugal machine, the control unit controls the centrifugal machine to centrifuge for 5-10 minutes at the speed of 400-800 revolutions per minute, the mechanical hand is used for taking out the culture flask with the cells, the screwing device is controlled to open the bottle cap, the supernatant is discarded, a proper amount of culture medium is absorbed for re-suspending the precipitated primary stem cells, and after the cell concentration is calculated under a microscope, the liquid transferring gun absorbs the quantitative complete culture medium to adjust the re-suspended cells to a proper concentration;
(8) After the control unit controls the inkjet marking device to mark the culture bottle obtained in the step (7), the manipulator places the marked culture bottle into CO 2 Culturing in a constant temperature cell incubator;
(9) The culture medium is replaced at 45-51 hours of culture interval of the culture bottle;
(10) When the growth proportion of the cells reaches 75% -90% through observation of a microscope, carrying out passage amplification culture;
(11) Centrifuging a culture bottle after passage expansion culture, discarding supernatant, adding PBS buffer solution to moisten and wash adherent cells, adding trypsin solution, putting the cells into a cell incubator to digest for 1-3min, stopping the cells in time with a complete culture medium when the cells are completely digested, centrifuging, discarding supernatant, adding frozen stock solution to a frozen stock tube, marking the frozen stock tube by an ink-jet marking device, putting the frozen stock tube into a program frozen stock device program, freezing to below-20 ℃, and transferring the frozen stock tube to a liquid nitrogen tank for storage;
in the step (9), a control unit controls a liquid transferring gun to absorb PBS buffer solution with medium quality in a culture bottle to the other culture bottle, after a screwing device is controlled to screw a bottle cap of the culture bottle, the cell culture bottle and a buffer solution culture bottle manipulator are moved into a centrifugal machine, the control unit controls the centrifugal machine to centrifuge for 5-10 minutes at the speed of 400-800 revolutions per minute, the bottle cap of the cell placing culture bottle is removed by the manipulator, the bottle cap is opened by the bottle controlling screwing device, a proper amount of culture medium is absorbed for re-suspending precipitated stem cells, and after the cell concentration is calculated by a blood cell counting plate under a microscope, the liquid transferring gun absorbs the quantitative complete culture medium to adjust the re-suspended cells to a proper concentration;
in the step (10), the control unit controls trypsin solution in the trypsin liquid storage tank to be resuscitated at a constant temperature of 37 ℃ in the trypsin solution transfer container; centrifuging the culture bottle containing the cultured cells after culturing in the step (9) to remove supernatant, soaking and washing the cells with PBS buffer solution, then, sucking trypsin solution into the culture bottle containing the cultured cells by a liquid-transferring gun, covering a cover, putting into a cell incubator, digesting for 1-3min, taking out the culture bottle, sucking a proper amount of complete culture medium in a complete culture medium transfer container into the culture bottle by the liquid-transferring gun, blowing the adherent cells remained on the surface of the bottle into the culture medium by a liquid-transferring gun, sucking PBS buffer solution with equal mass in a PSB buffer solution transfer container into another culture bottle, controlling a screwing device to screw the bottle cover by a control unit, then, moving the bottle cover into a centrifuge by a manipulator, controlling the centrifuge to centrifuge at a speed of 400-800 rpm for 5-10 min, taking out the culture bottle containing the cells by the manipulator, and controlling a screwing device to blow the culture bottle containing the cells by the manipulatorRemoving supernatant after opening the bottle cap, sucking complete culture medium into a culture bottle for precipitating cells, completely mixing the cells into single cell suspension, calculating cell concentration under microscope with a blood cell counting plate, adjusting cell suspension to proper concentration, subpackaging in the culture bottle, and placing the culture bottle into CO after marking the culture bottle by a control unit control inkjet marking device 2 And carrying out subculture in a constant temperature cell incubator at a ratio of 1:1.5-1:3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310829461.9A CN116855380B (en) | 2023-07-06 | 2023-07-06 | Stem cell expansion culture system and expansion culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310829461.9A CN116855380B (en) | 2023-07-06 | 2023-07-06 | Stem cell expansion culture system and expansion culture method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116855380A CN116855380A (en) | 2023-10-10 |
| CN116855380B true CN116855380B (en) | 2024-03-29 |
Family
ID=88222969
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310829461.9A Active CN116855380B (en) | 2023-07-06 | 2023-07-06 | Stem cell expansion culture system and expansion culture method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116855380B (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103013913A (en) * | 2012-11-30 | 2013-04-03 | 陆华 | Method for culturing human amniotic mesenchymal stem cells by enzyme digestion and EGF (epidermal growth factor) |
| CN107686811A (en) * | 2016-08-04 | 2018-02-13 | 发那科株式会社 | Manufacture system, information management system, conveying and the freezen protective equipment of stem cell |
| CN108102914A (en) * | 2017-12-27 | 2018-06-01 | 深圳北科赛动生物机器人有限公司 | Stem cell auto culturing system |
| CN109439545A (en) * | 2018-11-07 | 2019-03-08 | 深圳大学 | Biomedicine experiment equipment |
| CN112280737A (en) * | 2020-11-03 | 2021-01-29 | 威海见生生物技术有限公司 | Method for in vitro induced amplification and cryopreservation of immune cells |
| CN113528341A (en) * | 2021-06-03 | 2021-10-22 | 杭州捷诺飞生物科技股份有限公司 | Biological tissue production system and production method |
| CN216639513U (en) * | 2021-08-18 | 2022-05-31 | 英诺维尔智能科技（苏州）有限公司 | GMP (good manufacturing practice) compliance high-flux intelligent biological medicine production system |
| CN115261208A (en) * | 2022-09-09 | 2022-11-01 | 上海大学 | Full-automatic cell culture system and method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2017282153B2 (en) * | 2016-06-20 | 2022-08-04 | Genesis Technologies Limited | Automated cell processing systems and methods |
-
2023
- 2023-07-06 CN CN202310829461.9A patent/CN116855380B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103013913A (en) * | 2012-11-30 | 2013-04-03 | 陆华 | Method for culturing human amniotic mesenchymal stem cells by enzyme digestion and EGF (epidermal growth factor) |
| CN107686811A (en) * | 2016-08-04 | 2018-02-13 | 发那科株式会社 | Manufacture system, information management system, conveying and the freezen protective equipment of stem cell |
| CN108102914A (en) * | 2017-12-27 | 2018-06-01 | 深圳北科赛动生物机器人有限公司 | Stem cell auto culturing system |
| CN109439545A (en) * | 2018-11-07 | 2019-03-08 | 深圳大学 | Biomedicine experiment equipment |
| CN112280737A (en) * | 2020-11-03 | 2021-01-29 | 威海见生生物技术有限公司 | Method for in vitro induced amplification and cryopreservation of immune cells |
| CN113528341A (en) * | 2021-06-03 | 2021-10-22 | 杭州捷诺飞生物科技股份有限公司 | Biological tissue production system and production method |
| CN216639513U (en) * | 2021-08-18 | 2022-05-31 | 英诺维尔智能科技（苏州）有限公司 | GMP (good manufacturing practice) compliance high-flux intelligent biological medicine production system |
| CN115261208A (en) * | 2022-09-09 | 2022-11-01 | 上海大学 | Full-automatic cell culture system and method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116855380A (en) | 2023-10-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109661582A (en) | Automatic cytological processing system and method | |
| CN113528341B (en) | Biological tissue production system and production method | |
| CN116855380B (en) | Stem cell expansion culture system and expansion culture method | |
| CN106244455B (en) | Fully-automatic intelligent cell culture apparatus and its control method | |
| JP2004528020A (en) | Incubator and incubation method considering the organism to be incubated | |
| CN106701576A (en) | Fully-automatic cell culture room and control method thereof | |
| US5016688A (en) | Roller bottle filling and harvesting system | |
| JP2005304303A (en) | Supply and exhaust robot and automatic apparatus for culture | |
| JP2005034142A (en) | Culture treatment apparatus and automatic culture apparatus | |
| JP2005333823A (en) | Adapter for culture vessel and culture treatment apparatus | |
| JP2006141265A (en) | Cover for culture container, culture container and apparatus for culture treatment | |
| CN115261208A (en) | Full-automatic cell culture system and method thereof | |
| JP2005287466A (en) | Culture treatment apparatus | |
| CN209137462U (en) | A kind of bottle base sterilizing unit | |
| CN215668049U (en) | Biological tissue production system | |
| CN1404358A (en) | Liquid spawn inoculation device | |
| CN113549553A (en) | GMP (good manufacturing practice) compliance high-flux intelligent biological medicine production system | |
| CN202744548U (en) | Horizontal solid fermentation tank | |
| JP2005052069A (en) | Culture treatment apparatus and automatic culture apparatus | |
| CN214546164U (en) | Culture medium inoculation mixing arrangement | |
| JP2016208866A (en) | Automatic culture apparatus | |
| CN205740993U (en) | A kind of microorganism fermentation tank | |
| JP2006167496A (en) | Liquid feeding/discharging apparatus and culturing apparatus | |
| CN201334476Y (en) | Fermentation tank discharging device used for L-lactic acid production by Rhizopus oryzae semi-continuous fermentation | |
| CN111795553A (en) | Anti-pollution dehydration auxiliary device and application method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |